Biochemistry

Growing fatty acid chain step 3 and 4 - fatty acid biotransformation

3 Elimination: - Elimination of H2O form a double bond 4 Reduction - The double bond is reduced forming the saturated fatty acid group

Biosynthesis of sphingolipids -step 3 and 4

3 Formation of N-acylsphinganine (Dihydroceramide) - Attachment of fatty acid by Dihydroceramide synthase 4 Desaturation to form N-acylspingosine (ceramide)

Energetic cost of urea synthesis

4 high-energy phosphate groups are used for the synthesis of one molecule of urea - Two ATP for the formation of carbamoyl phosphate - One ATP is consumed to make arginosuccinate - A pyrophosphate cleavage to AMP and PPi Urea cycle cause - Oxaloacetate to fumarate (via aspartate) - Regeneration of oxaloacetate produce NADH - Each NADH can generate up to 2.5 ATP during mitochondrial respiration - Net is the use of 1.5 ATP for the synthesis of one molecule urea 2 NH4+ + HCO3- + 3 ATP4- + H2O --> Urea + 2ADP3- + 4Pi2- + AMP2- + 2H+

Cell signaling

A signal molecule binds to a receptor protein, causing it to change shape - The binding between signal molecule (ligand) And receptor is highly specific. - A conformational change in a receptor is often the initial transduction of the signal

Catabolic pathways for phenylalanine and tyrosine 2

Both phenylalanine and tyrosine is converted into - Fumarate and Acetoacetate and further to acetoacetyl-CoA Tyrosine is precursor of dopamine (neurotransmitter) and of morepinephrine and epinephrine Melanine is derived from tyrosine

Maintaining high driving force by hydrolysis of ATP

Both resonance of Pi and ionization of ADP keeps the driving force of ATP hydrolysis high

Processing of dietary fats

Chylomicrones with apolipoprotein CII move from intestinal mucosa and carried fatty acids to muscle and adipose tissue At the target tissue: Lipoprotein lipases are activated by apolipoprotein CII and degrade triacylglycerols to free fatty acids glycerol The lipase products are taken up by the cells: - In muscle: Fatty acids are oxidized for energy - In adipose tissue: reesterified to triacylglycerol for storage

Free radical reations

Compounds with an unpaired electron 'steals' an H atom - The compound comes from homolytic cleavage. - Usually induces an internal reaction in the substrate. - Often combined with other reaction types.

Condensation step 1 in fatty acid synthesis

Condensation of the activated acetyl and malonyl groups Formation of acetoacetyl-ACP The product is bound to ACP CO2 is released Catalyzed by the β-ketoacyl-ACP synthase (KS) The CO2 released in this reaction is the same as introduced by the acetyl-CoA carboxylase reaction Decarboxylation facilitate the nucleophillic attack

Glutamine synthetase (ATP enzyme) - all organisms

Strict, complex regulation: Allosteric Amino acids (A, G, H, W) AMP,CTP Carbamoyl P, GlcN-6-P Covalent: Adenylation of enzyme at Tyr397 Controlled by Gln

Serpentine receptor (G-receptor)

External ligand binding to receptor activates an intracelluar GTP-binding protein, which regulates an enzyme that generates an intracellular second messenger.

ATP Synthase has two functional domains FO and F1

F type ATPase, meaning that is really a synthase rather than an ATPase similar structure for mitochondria, chromoplasts, and eubacteria H+ flows from P side to N side (down gradient) ADP + Pi → ATP 2 main structural components FO integral membrane protein - F-O (not F zero) - O stands for oligomycin sensitive - If put into a membrane alone, the membrane leaks protons F1 peripheral protein - When isolated is an ATPase - Only when combined with FO and a properly oriented - H+ gradient does turn it into an ATP synthase

Each β subunit of ATP synthase can Assume Three conformations

F1 is α3β3γδε αβ form 3 sets of structures, like section of an orange ATP binding site is at αβ interface, but mostly on β γ is a central shaft that extends down into the FO δε is not visible in X-ray coordinates In X-ray observe 3 physical state of αβ (conformations): - one with ATP bound, - one with ADP and Pi bound and - one with nothing bound All three present at any one time

Formation of ether lipids - 3

Head group attachment - Essential like those in esther-linked phospholipids Introduction of double bound - Mixed function oxidase The synthesis take place in the peroxisomes

Mitochondria in thermogenesis, steroid synthesis and apoptosis

Mitochoandria has other functions than just making ATP Generates heat in adipose tissue Make steroids in adrenal glands and gonads - Lipid biosynthesis is otherwise made in the cytosol In most or all tissues key participant in programmed cell death (apoptosis)

Ion channels

Ion channels perform work by using established chemical and electrical gradients. - Voltage gated and ligand gated ion channels is essential for neural signaling

Thiamine Pyrophosphate Citric acid cycle

TTP is important in decarboxylations - Was introduced in the pyruvate -> ethanol conversion

Citric acid cycle overview

- Acetate enters (2 carbons) - 2 CO2 released - 3 NADH produced - 1 FADH2 produced - 1 GTP produced Acetyl CoA + 3NAD+ + FAD + GDP + Pi + 2H2O --> 2CO2 + 3NADH + FADH2 + CoASH + GTP + 3H+

Biosynthesis of sphingolipids step 5

5 Attachment of a head-group - Cerebroside (glucose) - Sphingomyelin (choline) - Take place in Golgi apperatus

Amino acid transformations using pyridoxal phosphate - Transamination

A. Transamination

Glucose-alanine cycle

Alanine also plays a special role in transporting amino groups to the liver Glucose-alanine cycle Degradation of amino acids for fuel Transfer of the a-amino group to pyruvate forming alanine - alanine aminotransferase In liver, alanine aminotrans-ferase transfers the amino group to a-ketoglutarate

Pyruvate → Ethanol

Alcohol fermentation In some plants and microorganisms Two step process producing CO2 and NAD+ (again) Pyruvate decarboxylase and alcohol dehdrogenase - Cofactors: Mg2+ and TPP Not a simple reaction - TPP plays an important role Decarboxylase: removal of a CO2 group (released to the cytosol

Amino acid transformations using pyridoxal phosphate - Racemization

B. Racemization

Structure of fatty acid synthase in different organisms

Bacteria and plants: Intermediates are not leaving the protein complex Vertebrates: - A single large polypeptide (Mr 240.000) - Function as a dimer - head to tail Working independently

Use of FAD rather than NAD+ in Succinate Oxidation

Based on the difference in standard reduction potential (ΔE'O) for each pair of half-reactions, one can calculate ΔG'O. The oxidation of succinate by FAD is favored by the negative standard free-energy change (ΔG'O = -3.7 kJ/mol). Oxidation by NAD+ would require a large, positive, standard free-energy change (ΔG'O = 68 kJ/mol).

Absorption of fatty acids in small intestine

Bile acids (e.g. taurocholate and glycolate) solubilize the dietary fat. Amphiphatic molecules Synthesized in liver from cholesterol Stored in the gallbladder Released into the small intestine after ingestion of a fatty meal.

Amino acid transformations using pyridoxal phosphate - Decarboxylation

C. Decarboxylation

Non-oxidative recycling phase carbon group transfers (pentose phosphate pathway)

Carbon group transfers of transketolase and transaldolase 6xRibose 5-phosphate → 5xGlucose 6-phosphate

Heteroplasm in mitochondrial genome

Different degree of heteroplasm in muscle tissue with defect cytochrome oxidase

The b-oxidation is repeated

During one pass of the b-oxidation: - 1x acetyl-CoA, - 2x pairs of electrons, - 4 protons

The enzyme complex citric acid cycle - Dihydrolipoyl transacetylase E2 (green)

E2 has three functionally distinct domains - Core domain - Binding domain (connected to E1 and E3) - Lipoyl domain where lipoate is bound -Lipoate can reach the active sites of E1 and E3 -Number varies in species

Elimination (metabolism)

Electrons are rearranged but oxidation state is unaltered. Elimination is often a small group leaving while a double bond is formed.

Oxidative deamination

Glutamate dehydrogenase - An oxidative deamination - An allosteric enzyme with six identical subunits - Mr 330.000 - Mutation in the regulatory site cause an elevated levels of ammonia in the blood

Chemiosmotic coupling allows non-integral stoichiometries

How to calculate the P/O ratio? - How many P's for each O? Complicated as ATP is consumed by mitochondria as well Most experiments indicated - 3 P for an NADH and 2 P for an FAD - Expectation of integral values Now measure H+ fluxes instead - 1 NADH moved 10 H+ - 1 FAD only moved 6 H+ Have seen that ATP synthase itself uses 3H+ Will need additional H+ for ATP, ADP, and Pi transport So we end up with about 2.5 ATP/NADH and 1.5 ATP/FAD

Formation of glycerol 3-phosphate

How to generate enough glycerol 3-phosphate In adipose tissue during starvation - Glycolysis is reduced Glyceroneogenesis - a short version of gluconeogenesis Formation of glycerol 3-phosphate from pyruvate via dihydroxyacetone phosphat

Receptor enzyme

Ligand binding to extracellular domain stimulates enzyme activity in intracellular domain. - Figure is Guanylyl cyclases which generates the second messenger cGMP.

Apolipoproteins

Lipid-binding proteins Transport triacylglycerol, phospholipids, cholesterol and cholesteryl esters in the blod between organs. Different lipid/protein structures: - Chylomicrons - VLDL - LDL - HDL - VHDL

Oxidation of unsaturated fatty acids

Most double bonds in unsaturated fat are in cis configuration Enoyl-CoA hydratase can not act on these cis double bonds Example: Oleate (D9 18-carbon fatty acid): - formation of oleoyl-CoA - transport into mitochondria - three phases of fatty acid oxidation --> cis-D3-dodecenoyl-CoA (C-12) Enoyl-CoA isomerase: trans-D2-dodecenoyl-CoA (L-b-hydroxyacyl-CoA)

Synthesis of phosphatidylinositol

Play a central role in signal transduction eukaryotes

Organization of phospholipids

Polar head groups organize the phospholipids into the characteristic membrane bilayer structure

Enzyme-catalyzed transaminations

Removal of the a-amino group (transamination) Amino transferases / transaminases The a-amino group is transferred to the a-ketoglutarate (an a-keto acid) - Leaving back another a-keto acid The result is L-glutamate

Amino acid biosynthesis

The Erythrose 4-phosphate pathway is blocked by N-(phosphono methyl) glycin Tryptophan, phenylalanine and tyrosine as end products only happens in bacteria, fungi and plants

Membrane fluidity

depends on; - lipid composition - structure of hydrophobic tails (unsaturated) - temperature Increased fluidity leads to decreased membrane thickness

Formation of leucotriens

mangler

RAS(Mitogen activated protein kinase)

stimulate a serin/threonin Kinase that activate MAP-kinases

Removal of water molecules from hydrophilic solute

very energy demanding process.

Glycogen regulation - Epinephrine

Epinephrine and glucagon binds to a membrane receptor Activates GTP-binding protein that leads to cAMP formation cAMP starts an enzyme cascade - A catalyst activates a catalyst which activates a catalyst - Signal amplification The signal from one hormone results in the conversion of 10,000 molecules of glycogen to glucose 1-phosphate Muscle: glycolysis is initiated, producing ATP for contraction Liver: Gluconeogenesis is initiated, producing glucose to elevate blood sugar

Enzyme elasticity (epsilon)

Epsilon illustrates the change in the catalytic effect - Sensitivity to substrate, product or effector concentration - Illustrated in the slope of the activity curve in a Michaelis-Menten graph (not the actual value of the slope) When [S] << Km, then epsilon = 1 When [S] >> Km, then epsilon = 0 In cooperative allosteric effect (nH > 1) the epsilon can be larger than one (but smaller than nH)

nonessentiel and essentiel amino acids for humans and the albino rat

Essential amino acids must be provided in the diet In meat and dairy products Alternative, in cereal grains + legumes

Regulation of fatty acid biosynthesis

Excess of energy is converted in to fatty acids - Stored as triglycerids Acetyl-CoA carboxylase is rate limiting - Inhibited by palmitoyl-CoA - Citrate is an allosteric activator, increasing Vmax - High citrate and ATP in mitochondria will activate the acetyl-CoA carboxylase - Citrate inhibits the activity of phosphofructokinase-1 Acetyl-CoA carboxylase is phosphorylated - Inactivation or reduced activity - Triggered by glucagon and ephinephrine Regulation at the level of enzyme expression - Intake of polyunsaturated fatty acids cause down-regulation of lipogenic enzymes in the liver

Mutase reaction - B12 coenzyme formation

Exchange of the group -CO-S-CoA at C-2 with a hydrogen atom at C-3 Coenzyme B12 - dependent reactions No mixing of the transferred hydrogen atom with a hydrogen atom from the solvent H2O

Excretory forms of nitrogen

Excretion of excess of NH4+ depends on the organism The carbon atoms of urea and uric acid are highly oxidized The organism discards carbon only after extracting most of its available energy of oxidation

Uncoupled mitochondria in brown fat - The real 'Fat Burner'

'Brown fat' is the single case where normal control of electron transport chain is subverted - Found in newborn animal and in hibernating animals - brown fat is brown because has unusually large amounts of mitochondria in fat cells The brown adipocytes have a protein called thermogenin - Also named uncoupling protein 1, (UCP1) Thermogenin allows H+ to flow back into mitochondria - without generating ATP Thus FA degradation gets uncoupled, and literally burn the fat to make heat!

Enzymes and cofactors of the nitrogenase complex - protein with ribbons

(a) In this ribbon diagram, the dinitrogenase subunits are shown in gray and pink, the dinitrogenase reductase subunits in blue and green. The bound ADP is red. Note the 4Fe-4S complex (Fe atoms orange, S atoms yellow) and the iron-molybdenum cofactor (Mo black, homocitrate light gray). The P clusters (bridged pairs of 4Fe-4S complexes) are also shown.

Nitrate assimilation by nitrate reductase and nitrite reductase.

(a) Nitrate reductases of plants and bacteria catalyze the two-electron reduction of NO3- to NO2-, in which a novel Mo-containing cofactor plays a central role. NADH is the electron donor. (b) Nitrite reductase converts the product of nitrate reductase into in a six-electron, eight-proton transfer process in which the metallic center in siroheme carries electrons, and the carboxyl groups of siroheme may donate protons. The initial source of electrons is reduced ferredoxin.

Enzymes and cofactors of the nitrogenase complex - protein with energy cloud

(a) The holoenzyme consists of two identical dinitrogenase reductase molecules (green), each with a 4Fe-4S redox center and binding sites for two ATP, and two identical dinitroge-nase heterodimers (purple and blue), each with a P cluster (Fe-S center) and an FeMo cofactor. In this structure, ADP is bound in the ATP site, to make the crystal more stable. (b) The electron-transfer cofactors. A P cluster is shown here in its reduced (top) and oxidized (middle) forms. The FeMo cofactor (bottom) has a Mo atom with three S ligands, a His ligand, and two oxygen ligands from a molecule of homocitrate. In some organisms, the Mo atom is replaced with a vanadium atom. (Fe is shown in orange, S in yellow.)

The two parts of metabolism

- Catabolic reactions Degradative - obtain building blocks and energy ATP, NADH and NADPH stores chemical energy and is often used as synonymes for energy - Anabolic reactions Biosynthesis - build up and growth (requires energy)

Composition of lipids and proteins

- Cholesterol may increase or decrease fluidity depending on other factors, such as the fatty acid composition of the other lipids found in the membrane. Decrease the permeability of lipidmembranes. - For any given membrane, fluidity also decreases with declining temperature. The membranes of cells that live at low temperatures tend to be high in unsaturated and short-chain fatty acids.

Laws of thermodynamics

- First law: Energy of the universe is constant; Using energy is just changing it to another kind E.g. Chemical Energy -> Heat, Heat -> Pressure, Pressure -> Work - Second law: The universe tends towards increased disorder Earphone cord; The disorder/randomness is called entropy (S) (quantification of the disorder)

Three main classes of membrane lipids

- Glycerolipids based on glycerol (see figure, part a; blue shading) with two C16-C18 fatty-acid chains (pink shading) linked at sn-1 and -2 (which forms diacylglycerol (DAG)). A cis-double bond is usually present in the fatty acid that is linked at sn-2, which causes a kink in the acyl chain and decreases the packing density of the lipid. A phosphate (P) can be attached at sn-3 (which forms phosphatidic acid (PA)) and this phosphate can carry a head group (R) that is either neutral (which produces phosphatidylserine (PS) or phosphatidylinositol (PI)) and gives a net acidic charge, or basic (which forms phosphatidylethanolamine (PE) or phosphatidylcholine (PC)) and gives a neutral or zwitterionic lipid. Sphingolipids are based on a C18 sphingoid base, which is usually sphingosine in animals (see figure, part b; blue shading), with a saturated C16-C26 fatty acid (pink shading) that is amide linked to the nitrogen (which forms ceramide (Cer)). In dihydroceramide, sphinganine replaces sphingosine (sphinganine lacks the trans-double bond between C4 and C5). Phytoceramide in plants and fungi contains phytosphingosine (C4-OH sphinganine) and a fatty acid that is usually hydroxylated at C2 (not shown). In animals, the sphingolipid head group (R in the left panel of part b of the figure) can be phosphocholine (which produces sphingomyelin (SM)) or phosphoethanolamine (to form ethanolaminephosphoryl ceramide (EPC)). Alternatively, in the case of glycosphingolipids, it can be glucose (to form glucosylceramide (GlcCer)) or galactose (to produce galactosylceramide (GalCer)), which can be further decorated by extra monosaccharides (all labelled G in the right panel of part b of the figure) to create a wide range of glycosphingolipids (for example, GM3). Plant and fungal sphingolipids typically contain a phosphoinositol head group (which forms inositolphosphorylceramide (IPC)) that is often mannosylated. Sterols are based on a planar four-ring structure (see figure, part c), with cholesterol being the form that is present in mammals, ergosterol in fungi, and stigmasterol and sitosterol in plants.

Cell membrane lipid componentand syntheses

- Most of the lipid molecules found in biological membranes are phospholipids. - Each has a hydrophilic region, where the phosphate groups are located, and a hydrophobic region, the fatty acid "tails." - The phospholipids organize themselves into a bilayer. - The interior of the membrane is fluid, which allows some molecules to move laterally in the membrane.

Glycolysis and Citric Acid Cycle energy

1 Glucose = 2 Pyruvates = 2 Cycles in CAC Glucose conversion in glycolysis: Glucose + 2NAD+ +2ADP + 2Pi → 2pyruvate + 2NADH + 2H+ +2ATP + 2H2O Pyruvate (Acetyl-CoA) conversion in CAC: 2Pyruvate + 8NAD+ + 2FAD + 2ADP + 2Pi + 4H2O → 6CO2 + 8NADH + 2FADH2 + 2ATP + 6H+ Total reaction: Glucose + 10NAD+ + 2FAD + 4ADP + 4Pi + 2H2O → 6CO2 + 10NADH + 2FADH2 + 4ATP + 8H+ We are getting close to the respiration formula; Glucose + 6O2 -> 6H2O + 6CO2 + Energy In the respiratory chain, electrons + O2 + H+ makes H2O + ATP (energy)

beta-oxidation of saturated fatty acids

1 step: Dehydrogenation of fatty acyl-CoA - Formation of double bond between the alpha and beta carbon atom trans-delta^2-enoyl-CoA Acyl-CoA dehydrogenase (ACD) Isoforms of acyl-CoA dehydrogenase: - Long-chain ACD: C12 to C18 fatty acids - Medium-chain ACD: C4 to C14 fatty acids - Short-chain ACD: C4 to C8 fatty acids Electrons transferred to FAD and further to electron-transferring flavoprotein in electron transporting chain Step 2 Addition of water to the double bond - Formation of the L-stereoisomer of b-hydroxy-acyl-CoA Also known as 3-hydroxyacyl acyl-CoA Catalyzed by enoyl-CoA hydratase Analogous to the fumarase reaction of citric acid cycle Step 3 Dehydrogenation of L-b-hydroxyacyl-CoA - Formation of b-ketoacyl-CoA Catalyzed by b-hydroxyacyl-CoA dehydrogenase NAD+ is the electron acceptor The enzyme is specific for the L-stereoisomer of hydroxyacyl-CoA Analogous to the malate dehydrogenase reaction of citric acid cycle Step 4 Split off the two carboxy-terminal two-carbon fragment - Formation of acetyl-CoA and acyl-CoA Catalyzed by acyl-CoA acetyl transferase (thiolase) Overall: The single bond between methylene groups in fatty acids is stable - The three first steps of the beta-oxidation create a much less stable C-C bond - C2 is now surrounded by two carbonyls - C3 is the target for a nucleophilic attack

How to generate the Sequence of electron carriers

1. Based on the potentials (only a guess, as standard potential are for standard conditions only) 2. Based on experiments 1) Exhaust O2 supply so everything stops - And everything stuck in reduced form - Add O2 and watch each species become oxidized a) Quick oxidation at O2 end b) Slow oxidation at beginning 2) Inhibition - Certain substances can inhibit certain points in flow - Add inhibitor and see what thing get backed up in reduced form, and what still get oxidized

Proton-motive force

1. Difference in proton concentration - Chemical potential energy 2. Separation of charge - Electrical potential energy Free-energy change of an electrochemical gradient: ΔG=RT ln (Cout /Cin) + Z F Δψ ln (Cout /Cin) = 2.3 (log [H+]P - log [H+]N) = 2.3 (pHP - pHN) = 2.3 ΔpH ΔG = RT ln (Cout /Cin) + Z F Δψ ΔG = RT ΔpH + Z F Δψ ΔG = (5.70 kJ/mol) ΔpH + (96.5 kJ/V * mol) Δψ In active mitochondria, proton Cout is 0.75 pH units lower (H+ higher) than the matrix (Cin) Δψ is about 0.15 - 0.2 V (outside positive - inside negative) --> Net E about 20 kJ/proton - NADH transported 10 proton out so this is - 10 * 20 = 200 kJ of E So most of the E is stored in potential gradient All we have to do is let the proton slide back in to get the energy back

Absorbtion of dietary fats

1. Forming mixed micelles of bile salts and triacylglycerols - increase the fraction of lipids accessible to the water soluble lipases 2. Lipases degrade triacylglycerols to - monoacylglycerols - diacylglycerols - free fatty acids - glycerol 3. Diffusion of lipase products into the epithelial cells 4. Formation of Cholymicrons (lipoprotein aggregate) from - triacylglycerol - dietary cholestrol - proteins

The 10 regulatory steps in enzyme regulation

1: Change of concentration of a extracellular signal, starting a signal pathway in the cell - Binding membrane receptor or enters the cell 2: Change of gene transcription by transcription factors - Several genes can be regulated by one transcription factor 3:Difference in mRNA stability - Fewer proteins gets translated from the mRNA if the lifetime is short 4: Difference in translation speed 5: The rate of enzyme degradation time varies From minutes to days 6: Enzymes can be isolated in cellular compartments - Step 1-6 regulates the number of enzymes in the cell (quantitative control) 7: Enzyme activity depends strongly on substrate concentration - Substrate concentration can be regulated (other enzymes) 8: Allosteric effectors are important regulators - Binds to enzymes and changes their structure - Can lead to both an enhanced or reduced activity 9: Covalent modifications of the protein - Often (de)phosphorylation - Is together with allosteric regulation the most important mechanisms 10: Binding of regulatory proteins - Quite the same mechanism as allosteric effector - Step 7-10 regulates the activity of each enzyme (qualitative control)

Ubiquinone - in oxidative phosphorylation

A benzoquinone with a long isoprenoid tail Diffuses freely in membrane Can accept 1 or 2 e^- electrons: Q ↔ QH ↔ QH2. Carries both electrons and protons H^+ flow across membrane Closely related to plastoquinone in plants

The triacylglycerol cycle

A constant recycling of triacylglycerols in adipose tissue Exchange with other tissues Low exchange between liver and adipose tissue when other fuels are present About 75% of the released fatty acids in adipose tissue are re-esterified

Activation energy

A large energy barrier sometimes prevents otherwise spontaneous reactions from occurring - Very slow conversion rate Enzymes provides alternative pathways, catalyzing the reaction - The enzymes do not change the free energy nor the equilibrium constant of the reaction, only the rate of conversion. Finally, the deltaG'^o of sequential reactions can be added to yield the deltaG'^o of the total reaction A -> B, deltaG'^o =-15 kJ/mol B -> C, deltaG'^o =5 kJ/mol A -> C, deltaG'^o =-15+5=-10 kJ/mol

N-Acetyl Glutamate Synthetase Deficiency (NAGS)

A rare genetic disorder characterized Complete or partial lack of the enzyme NAGS Lack of the NAGS enzyme results in excessive accumulation of nitrogen, as ammonia in the blood (hyperammonemia) Ammonia is a neurotoxin Symptoms of NAGS include - vomiting - refusal to eat - progressive lethargy - coma. NAGS deficiency is inherited as an autosomal recessive trait.

Insulin receptor

A tyrosine specific protein kinase

Mechanisms of adipocyte differentiation

A) Differentiation of mesenchymal stem cells (MSCs) and effect of angiotensin II type-1 receptor blocker (ARB) on this adipogenesis. In differentiated adipocytes, secretion of inflammatory adipocytokines, such as TNF-α, is decreased, and beneficial cytokines, such as adiponectin, are increased, whereas the opposite is observed in MSCs. Treatment with an ARB inhibits adipogenesis. Such treated adipocytes may transdifferentiate into other types of cells. B) Differentiation of preadipocytes and effect of ARB on this adipogenesis. Preadipocytes treated with an ARB differentiate into small adipocytes and secrete decreased inflammatory cytokines, such as TNF-α, and increased beneficial cytokines, such as adiponectin, and are regarded as well differentiated adipocytes. In contrast, lack of RAS blockade has the opposite effect on secretion in large adipocytes, regarded as poorly differentiated adipocytes. ARB: angiotensin II type-1 receptor blocker

Phosphoryl transfer and ATP

ATP is synthesized during catabolism - Yield from energy rich nutrients - Storage of chemical energy ATP is used during anabolism; Helps drive the endergonic processes - Synthesis of macromolecules - Obtaining cellular gradient - Muscle contractions Phosphate of ATP is hydrolyzed - Large negative deltaG'^o - deltaG is usually even larger (negative) - Much energy can be harnessed - Often Mg2+ is involved in phosphoryl group transfers - Partially shields negative charges and influence the conformation of phosphate groups (ATP and ADP) ATP levels are kept high in cells - Pi is kept low - assisted by resonance - Far from equilibrium - Keeps a high driving force - The high potency (low deltaG) of ATP is maintained

ATP in endergonic reactions

ATP helps drive the endergonic processes - Glutamate + NH3 -> Glutamine + H2O - deltaG'^o = 14.2 kJ/mol - ATP + H2O -> ADP + Pi - deltaG'^o = -30.5 kJ/mol - Combined: ATP + Glutamate + NH3 -> Glutamine + ADP + Pi - deltaG'^o = -16.3 kJ/mol The reactive phosphoryl is added to glutamate and then exchanged for ammonia (both exergonic)

ATP is stabilized relative to ADP on the surface of F1

ATP hydrolysed by F1 in the presence of 18O water The Pi formed does contain 3 to 4 18O This exchange occur without energy suplus ΔG'º for ATP synthesis and hydrolysis on the enzyme is close to zero ΔG'º for ATP in solution is - 30.5 kJ/mol When bound to F1, ATP is hydrolyzed and resynthesized rapidly. ADP + Pi W ↔ ATP is in equilibrium ATP synthase stabilizes relative to ADP + Pi ATP is bund more tightly - FOF1 binds ATP with high affinity (Kd ≤ 10-12 M) - FOF1 binds ADP with lower affinity (Kd ≈ 10-5 M) Drives the equlibrium toward formation of ATP ΔG of the reaction is not changed! - The principles of enzyme catalysis is that the ΔG of a reaction is not changed - The ATP formation is on the surface of the enzyme, not yet back in solutions The ΔG of the reaction is not changed because the reaction is not complete. - The ADP and ATP are in equilibrium on the enzyme, - a big push of E is needed to get the tightly bound ATP of the enzyme again. This is where the proton gradient comes in. - It is going to give us the E push

Importance of the ATP/AMP ratio - enzyme regulation

ATP is an important mediator of many reactions due to the high negative ∆G The ratio between ATP and ADP has to be high to maintain this high negative free energy The regulatory mechanisms reacting to a drop in ATP are thus very important As ADP can be converted to AMP and AMP is found in very low concentrations, the relative change is largest for AMP and is thus used as the regulatory key When [AMP] rises, and [ATP ]drops, AMP-activated protein kinase (AMPK) is activated AMPK activates ATP-producing processes and inhibits ATP consuming processes

Recap of Phosphoryl group transfers and ATP

ATP is synthesized during catabolism and used during anabolism - Used for storing and transferring chemical energy - Transfer of phosphoryl groups releases much free energy - Phosphoryl/pyrophosphoryl/ adenylyl groups are transferred to compounds to facilitate their further reaction ATP concentration is kept high - Far above equilibrium - Keep the group transfer potential high Other compounds as thioesters and phosphoenolpyruvate have large negative free energies of hydrolysis as well Chains of inorganic phosphate are stored in all cells as a reservoir of phosphoryl groups High energy demanding cells as myocytes have phosphocreatine for a fast rebuild of ATP levels

Formation of Ketone Bodies

Acetyl-CoA - Enter citric acid cycle OR - converted to "ketone bodies" Ketone bodies - Acetone - Acetoacetate delta-beta-hydroxybutyrate Step 1: Formation of acetoacetate 1 Condensation of two acetyl-CoA - Catalyzed by thiolase - Reversal of the last step of beta- oxidation 2 Condensation with an additional acetyl-CoA - Formation of beta-hydroxy-beta-methylglutaryl-CoA (HMG-CoA) - Catalyzed by HMG-CoA synthase Step 2: Formation of acetoacetate 3. beta-hydroxy-beta-methylglutaryl-CoA (HMG-CoA) is cleaved to free acetoacetate and acetyl-CoA - Catalyzed by HMG-CoA lyase Reduction of acetoacetate to D-beta-hydroxybutyrate - Reversible process - Catalyzed by D-beta-hydroxybutyrate dehydrogenase, a mitochondrial enzyme Small amounts of acetone is formed in healthy individuals - In untreated diabetes, large quantities of acetoacetate is formed

Recap of the Reactions of the Citric Acid Cycle

Acetyl-CoA enters the CAC, and goes through its eight steps, producing: - 2 CO2, 3 NADH, 1 FADH2, 1 GTP and CoA-SH - In the first step it reacts with oxaloacetate, which is also the final product - it is recycled - Step 1, 3 and 4 are irreversible From one glucose we obtain 30-32 ATP molecules - only 5-7 are from the glycolysis Around 60 % of the chemical energy of glucose is harnessed Precursors for various anabolic reactions comes from the CAC - The Citric Acid Cycle needs to be 'fed' with intermediates to provide oxaloacetat for acetyl-CoA to react with to enter the cycle

Active transport and passive transport

Active transport: Against gradients. - If ATP is used directly for the pumping system, as in the sodium-potassium pump, the system is a primary active transport system. - Only cations, such as sodium, potassium, and calcium, are transported directly by pumps that use a primary active transport system. Passive transports: Down gradients

Structure of fatty acid synthase type I system

Acyl carrier protein (ACP) - Contain the prosthetic group: 4'phosphopantheine - Derived form the vitamin pantothen acid - Identical to the group found in CoA

To start the next cycle in fatty acid synthesis

Addition of malonyl group to the phosphopantetheine-SH group on ACP Condensation reaction with the butyryl group as with the acetyl group before The product is a six-carbon acyl group - Reduction of the carbonyl group - Dehydration - Reduction of double bond

Energy effectiveness of Citric Acid Cycle

After the CAC we get no more energy out of the carbon source (Glucose -> 6CO2) From one glucose we got in total 32 ATP - Hydrolysis of one ATP to ADP has an actual free energy of -52.5 kJ/mol We obtained a total of 32 * 52.5 kJ/mol = 1680 kJ/mol The theoretical maximum energy of glucose is 2840 kJ/mol - not all energy is harnessed 1680/2840 * 100 = 60 % of the chemical energy of glucose is obtained

Electron carriers in multienzyme Complex III

Also called Cytochrome bc1 complex or Ubiquinone:cytochrome c oxidoreductase Electrons from QH2 transferred to cytochrome c More vectorial transport of protons Complex III is a dimer of identical monomers - Each consist of 11 subunits - 250,000 MW - The functional core is three - subunits: 1. Cytochrome b and its two heme (heme bH and bL) 2. Reiske iron-sulfur protein with its 2Fe-2S centers 3. Cytchrome C with its heme Complex III have two binding sites for ubiquinone Net equation: QH2 + 2 cyt c1 (oxidized) + 2H+N → Q + 2 cyt c1(reduced) + 4H+P Cytochrome c is a 1-electron carrier So, 1 QH2 donates their electrons to two cyt C The Q cycle: - 1 electron is donated to 2Fe-2S complex (further to Cyt C) - The other electron reduce the quinone to a semiquinone radical - The next set of electrons are split in the same way forming QH2 - Two of the protons released on the P-side are electrogenic Cytochrome C is found in the intermembrane space

Electron carriers in multienzyme Complex IV

Also called cytochrome oxidase Carries electrons from cytochrome C to O2 and reducing it to H2O Large complex of 13 subunits, MW 204,000. Compare with bacterial complex IV which is much simpler, with three subunits - Three major subunits in mammalian, are subunit I, II and III Subunit I (Yellow) - 2 heme's: heme a and a3 - 1 cobber ion (CuB) Subunit II (Blue) - 2 Cu ions complexed by cys (called CuA) - Looks like an 2Fe-2S center Subunit III (Green) Electron passed from 4 cytochromes c to CuA (subunit II) From CuA → heme a → heme a3 → CuB subunit I From CuB → O2 Every 4 electrons makes 2H2O Using H+ from inside Also pumps 4 H+ from in to intermembrane space NET reation: 4 Cyt c (red) + 8H+N + O2 → 4 Cyt c (ox) + 4H+P + 2H2O Does this in four 1-electron steps Yet no intermediates like OH-, HO• or peroxide releases.

Points of entry into citric acid cycle

Amino acid catabolism account for only 10-15% of human body's energy production Five products of carbon skeleton of amino acids enters citric acid cycle - Acetyl-CoA (10 aa.) - a-ketoglutarate (5 aa.) - Succinyl-CoA (4 aa.) - Fumarate (2 aa.) - Oxaloacetate (2 aa.)

The anammox reactions.

Ammonia and ammonium hydroxide (hydroxyl amine) are converted to hydrazine and H2O by hydrazine hydrolase, and the hydrazine is oxidized by hydrazine-oxidizing enzyme, generating N2 and protons. The protons generate a proton gradient for ATP synthesis. On the anammoxosome exterior, protons are used by the nitrite-reducing enzyme, producing ammonium oxide and completing the cycle. All of the anammox enzymes are embedded in the anammoxosome membrane.

Glutamine transport of ammonia

Ammonia is quite toxic to animal tissue The free amonia is combined with γ-glutamyl phosphate to form glutamine - Glutamine synthetase; The reaction require ATP 1. First, g-glutamyl phosphate is formed 2. Next, g-glutamyl phosphate react with ammonia forming glutamine Glutamine serve as a amino group donor in various biosynthetic reactions

Response coefficient (R)

An expression of how sensitive the reaction which the enzyme catalyzes is to outer factors such as hormones - Not other enzymes, or metabolites R is a product of the flux coefficient and the elasticity R = C*epsilon It is thus dependent on the relative effect of the specific enzyme in a pathway and its sensitivity to changes The regulated enzymes can be divided in two groups - Control enzymes (alter the flux through the pathway) - Response to outside signals E.g. low blood sugar: start glycogen -> glucose - Regulatory enzymes (ensures homeostasis when flux is altered) - Hold each intermediate metabolite at a constant levels - 'ships' them through the pathway

Mitochondria are central to the initiation of Apoptosis

Apoptosis - controlled or programmed cell death - used when individual cells die for the good of the organism - When a stressor or a signal tell the cell it is time to die One early marker of apoptosis is that mitochondria release cytochrome c from intermembrane space into the cytosol.

Rotational catalysis in Fo

As H+ streams through core of FO, FO appears to rotate relative to a and b Rotation of c subunits of FO makes the γ part of F1 move γ of F1 interacts with each αβ dimer in F1, get conformational changes that accept ADP, and Pi, then force then into ATP, then change binding so releases ATP Each rotation of 120º takes 3H+ and generates 1 ATP from ADP and Pi One can see this motion in a fluorescent microscope if attach a fluorescent label to FO In fact saw rotate one direction when makes ATP and the other when is an ATPase

Recap of the protion of Acetyl-Coa (activated acetate) in the citric acid cycle.

At aerobic conditions pyruvate/acetyl-CoA goes through the CAC - Produces FADH2 and NADH that can be used in the respiratory chain to obtain ATP Glucose (or fatty/amino acids) catabolism has three stages; 1 Production of Acetyl-CoA (Glucose -> Acetyl-CoA) 2 Acetyl-CoA oxidation (CO2 + NADH/FADH2) 3 Electron transfer and oxidative phosphorylation (NADH/FADH2 + ADP + Pi + ½O2 -> NAD+/FAD + ATP + H2O) - Together: cellular respiration Pyruvate dehydrogenase (PDH) complex converts pyruvate and CoA into Acetyl-CoA in a complex reaction involving a total of five cofactors

Definition of a cell membrane

Bilayered dynamic structues that: - perfom vital physiological roles - form boundaries between cells and their environment - regulate movement of molecules into and out of the cells Lipids, proteins and carbohydrate in various combinations make these tasks possible. The area of the membrane is 1/10 of the total area of internal membranes.

Adhesion receptor

Binds molecules in extracellular matrix, changes conformation, thus altering its interaction with cytoskeleton

Formation of ether lipids - 2

Biosynthesis of - Plasmalogens - Platelet-activating factor Formation of an ether lipid Reduction to an alcohol Introduction of a fatty acyl group

Biosynthesis of fatty acids

Biosynthesis of fatty acid is not the reversal of the fatty acid oxidation Fatty acid breakdown and biosynthesis - are different pathways - catalyzed by different enzymes - take place in different parts of the cell Biosynthesis is based on the three-carbon intermediate - Malonyl-CoA

Phosphate Translocase

Brings H2PO4 in the cell (Pi) into the mitochondrial matrix The outside of membrane is positive (+), why bringing a negative ion (-) in would NOT be a favored Bring in an H+ with the Pi. - A symport process, to make it energetically feasible Net: takes 1 more H+ for every ATP synthesized

Metabolic flux coefficient (C)

C is used to quantify the metabolic flux of a pathway - The relative contribution of each enzyme to setting the flux (J) of metabolites Determination example - Homogenized liver tissue -3 first steps of glycolysis -Measurements of F1,6-BP Change of flux when adding more enzyme - Relative values are calculated (C) - Hexokinase IV: 0.79 - Phosphohexose isomerase: 0 - Phosphofructokinase-1: 0.21 C is between 0 and 1 (or negative: flux out of pathway - e.g. Pentose phosphate pathway) The sum of all C's involved in a pathway is 1 0.79 + 0.0 + 0.21 = 1.0

Two strategies for forming the phosphodiester bonds

CDP: Cytidine diphosphate

Entropy

Can be ascribed to both energy and matter (box 1.3) - Fx boiling water in a kettle; Energy: Boiling water in cold surroundings leads to divided energies and low entropy; Heat (energy) will pass to the surroundings. Even temperature in the 'universe'. Uniform or random distribution of temperature = low energy. Irreversible (water will never spontaneous start to boil) Matter: Molecules have a value of entropy themselves Reaction: C6H12O6 + 6O2 -> 6CO2 + 6H2O Increased number of molecules = higher entropy Also increased entropy at transition from solid to liquid or liquid to gas

Recap of Feeder pathway for glycolysis, and fates of pyruvate under anaerobic condition.

Carbohydrates of many different origins enter at different points of the glycolysis Pyruvate still contains much chemical energy Pyruvate has three pathways after glycolysis - conversion into: - Acetyl-CoA To the citric acid cycle - harness more energy Aerobic metabolism - Lactate Anaerobic metabolism (fermentation) Heavy muscle activity Produces NAD+ - helps drive the glycolysis but give no further energy - Ethanol Anaerobic metabolism (fermentation) Some plants and microorganisms Produces NAD+ as well

Breakage or formation of C-C bonds (metabolism)

Carbonyl group is important! The carbon is partial positive (electrophilic) and can delocalize charge, facilitating the formation of a carbanion on a neighboring carbon.

Pyruvate's pathway

Catabolism of fatty acids, amino acids and pyruvate yields acetyl-CoA - They can all go through the CAC - Amino and fatty acid catabolism to acetyl-CoA will come later Pyruvate can be converted to acetyl-CoA in one step - Pyruvate dehydrogenase complex (PDH)

Glycolysis step 2: Glucose 6-P -> Fructose 6-P

Changing ring structure by phosphohexose isomerase and Mg2+ - Isomerase: Changing isomers. One substrate into one product Active glutamate opens ring, abstracts a proton leading to bond reformation, a new proton is reinserted in a new position, closing at a different carbon and release Reversible

Formation of Coenzyme B12

Cleavage of triphosphate from ATP One additional reaction: formation of S-adenosylmethionine from methionine and ATP

Glycogen phosphorylase

Cleaves off one glucose residue from the nonreducing end of glycogen - Inorganic phosphor is added, yielding Glucose 1-phosphate - No ATP is spend - Stops four residues before a branching point (in the gut the linkage is broken by amylase by hydrolysis)

Electron carriers in multienzyme Complex I and II

Complex I: Also called NADH:unbiquinone oxidase The complex contain - 42 different polypeptide chains - 1 FMN flavoprotein - At least 6 Fe-S centers By electron microscopy one can see that complex I is L shaped One end sticks into inside of mitochondria The full chemical reaction is: - NADH + H+ (from matrix) + Q → QH2 (in membrane) + NAD+ (exergonic) - Transfer of 4 protons from matrix to intermembrane space The complex is a vectorial proton pump - Pumps protons in one direction Generate both a concentration gradient and a charge gradient The full reaction can be written as : NADH + 5H+N + Q → NAD+ QH2 + 4H+P Where H+N means protons on negative side of membrane (inside) And H+P means protons on the positive side (periplasmic space) Amytal, rotenone and piericidin A - inhibits the electron flow from Fe-S centers of Complex I to ubiquinone - Block the overall process of oxidative hosphorylation Complex II also known as Succinate dehydrogenase from the TCA cycle Smaller and simpler than complex I - At least 4 different proteins (A,B, C and D) - Five different prosthetic groups of two types Proteins - Subunits C and D are integral membrane proteins - Contain a heme b - Contain a binding site for ubiquinone - Subunits A and B extend into the matrix - Contain three Fe-S centers, a bound FAD, and a binding site for succinate Electron move from succinate → FAD → FeS centers → ubiquinone

Citric acid cycle - Lipoate

Contains two thiol groups that can create a disulfide bond (-S-S-) Can serve both as an electron and acyl carrier Bound to a lysine on E2 of the enzyme complex

Chemiosmotic coupling

Coupling between - ATP production and - flow through electron transport chain (= oxygen consumption)

Recap of Chemical Logic and Common Biochemical reactions

Covalent bonds can be broken in two different ways - Hetero- and homolytic At heterolytic cleavage one molecule either; - Gets both electrons and becomes a nucleophile - Gets no electrons and becomes an electrophile Electrophiles and nucleophile tend to react with each other Reactions are divided in 5 major groups - Breakage/formation of C-C bonds - Internal rearrangements and eliminations - Free-radical reactions - Group transfers - Oxidation-reductions The changes are all steps in the various metabolic pathways, controlling the faith of the compounds - Often facilitated by enzymes - Can be tightly regulated

Cleavage and bond formation (metabolism)

Covalent bonds can be cleaved in different ways; Homolytic - share of electrons; Heterolytic - one gets both electrons. Formation of bonds often involves a nucleophile and an electrophile; Nucleophile: wants nuclei and have excess electrons to donate; Electrophile: wants electrons. These can come from heterolytic cleavage

Electro-chemical gradients

Creates driving force for transport of charged molecules

Coenzyme A

Cytosolic Coenzyme A - Activation of fatty acids - Biosynthesis of fatty acids Mitochondrial Coenzyme A - Oxidative degeneration of pyruvate, fatty acids, amino acids

Diffusion rate

Diffusion over large distances is very slow. - Are determined by temperature, size of the molecule, electrical charge of the molecule, and concentration gradient. - The insertion of a biological membrane affects the movement of chemicals in solution according to the membrane's properties. It may be permeable to some molecules and impermeable to others.

Internal rearrangements (metabolism)

Electrons are rearranged but oxidation state is unaltered. Internal rearrangements moves double bonds around or cis/trans formations and is often driven by isomerases.

Hydrogen exchange - B12 coenzyme formation

Dissociation energy: Co3+- C5adenosin 110 kJ/mol C-C bond 348 kJ/mol C-H bond 414 kJ/mol 1. Formation of 5'adenosyl radical and Co2+ form 2. The radical extracts a hydrogen atom from the substrate forming a new radical 3. Rearrangement yields a second radical 4. The hydrogen atom is donated to the radical 5. The bond reforms between cobalt and the •CH2-. Vitamin B12 deficiency: - needed 3 µg/day - pernicious anemia (Malabsorbtion of Vitamin B12)

Glycogen synthesis

During high energy periods When glycogen is synthesized from monosaccharides the intermediate step is usually a sugar nucleotide - E.g. glucose 1-P has been transferred to UTP by the expense of 2 Pi: Glucose 1-P+UTP → UDP-Glucose + PPi (or 2Pi) This has several positive properties - The reaction is irreversible - The nucleotide has many reactive groups - The nucleotidyl is an excellent leaving group facilitating nucleophillic attack - The nucleotide can serve as a sorting-tag for specific purposes diverse from free hexoses (tagged for for glycogen synthesis instead of glycolysis) Glycogen synthase transfers the UDP-sugar to the nonreducing end of a glycogen chain - UDP is released - Chain has to be longer than four residues The glycogen branching enzyme makes branches - Transfers 6-7 residues to a more interior position on the same branch - Creates a alpha1→6 linkage The branching has two purposes - Increased solubility of the glycogen complex - More available nonreducing ends - More targets for fast build up or break down

Growing fatty acid chain step 1 and 2 - fatty acid biotransformation

Each malony group and acetyl group is activated by the thioester links 1 Condensation: - Elimination of CO2 - Extends the acyl chain by two carbons Reduction of the β-keto in three steps - As found in the β-oxidation 2 The β-keto group is reduced to an alcohol

The 5 steps in the metabolic pathway

Each step is usually facilitated by an enzyme. The major groups depends on the nature of the change on the substrate 1. Breakage or formation of C-C bonds 2. Internal rearrangements and eliminations 3. Free-radical reactions 4. Group transfers; Commonly an acyl, glycosyl or phosphoryl group 5. Oxidation-reductions

Oxidation-reduction reactions

Electron transfer between molecules - Key reaction in metabolism - Directly or indirectly responsible for all work done - Initial electron donors come from food (or species exited by the absorption of light) Electron donors donate electrons to species with high electron affinity - Energy is released - Proportional to the difference in electron affinity - Electromotive force - Same principle as batteries Shared electrons are not 'owned' equally - More electronegative atoms have higher affinity - Rank of electro negativity of common atoms: - H < C < S < N < O - When a double bond is broken the shared electrons tend to follow the most electronegative atom - The electronegativity increases from left to right in the periodic system.

Ways of electron transfer

Electron transfer is divided into four groups - One conjugate redox pair transfer an electron to another pair (e.g. the Fe/Cu example) - Via hydrogen atoms (AH2 + B ↔ A + 2e- + 2H+ + B ↔ A + BH2) - As a hydride ion (:H-, has two electrons) - Through direct combination with oxygen (electron acceptor) - R-CH3 + ½O2 → R-CH2-OH

Recap of Biological Oxidation-Reduction reactions

Electrons are transferred between molecules - Very important in metabolism - Energy can be obtained - Driven by a difference in electron affinity A species that donates an electron is called a reductant. The species receiving them are called oxidants Reduction potentials is the quantification of the driving force of electron transfer between species - At standard conditions the standard reduction potential (E'°) can be measured - When knowing the concentrations of the electron donor and acceptor, the actual reduction potential (E) can be calculated - From deltaE (or deltaE'°) deltaG (or deltaG'°) can be calculated The electron carriers NAD, NADP, FAD and FMN play an important role in mediating the electron transfer (reduction and oxidation)

Nitrogen fixation by the nitrogenase complex.

Electrons are transferred from pyruvate to dinitrogenase via ferredoxin (or flavodoxin) and dinitrogenase reductase. Dinitrogenase reductase reduces dinitrogenase one electron at a time, with at least six electrons required to fix one molecule of N2. An additional two electrons are used to reduce 2 H+ to H2 in a process that obligatorily accompanies nitrogen fixation in anaerobes, making a total of eight electrons required per N2 molecule. Binding of ATP shifts the reduction potential from -300 mV to -420 mV

Glycolysis step 9: 2-Phosphoglycerate → Phosphoenolpyruvate (PEP)

Enolase dehydrates 2-Phosphoglycerate to PEP in a reversible reaction PEP has a much higher phosphoryl group transfer potential Reversible

The five consecutive reactions of PDH - The enzyme complex in the citric acid cycle

Enzymes and coenzymes are clustered - Intermediates never leave the complex - Fast reaction - Called substrate channeling

Preventive effect of aspirin

Epidemiological studies: - Reduce fever and pain - Reduce the risk of heart attack - Reduce the risk of development of colon cancer

Conformation of FO

FO has 3 subunits ab2c10-12 C is small, 8000 AA very hydrophobic, 2 membrane spanning helixes and a loop on the matrix side Attached to F1 by γε so F1 stand on top of FO cylinder a and b are to the side and anchor to δ on side of F1

Gluconeogenesis bypassing step 3 of glycolysis

Facilitated by Fructose 1,6-Bisphosphatase (FBPase-1) + Mg2+: Fructose 1,6-bisphosphate + H2O → Fructose 6-phosphate + Pi Inorganic phosphate is released, and not transferred to form ATP Irreversible

Pyruvate → Acetyl CoA

Facilitated by pyruvate dehydrogenase complex (PDH) Cofactors are TPP, lipoate and FAD Addition of CoA Decarboxlylation and oxidation Aerobic metabolism (further in the pathway) - Acetyl-CoA is further oxidized to CO2 in the citric acid cycle (to come) - Production of ATP

Biosynthesis of triglycerides

Fatty acids - Incorporation in triacylglycerols (storage) - Incorporation in phospholipids (membranes) Precursors - L-glycerol 3-phosphate - Fatty acyl CoA Formed from dihydroxyacetone phosphate - Glycerol 3-phosphate dehydrogense Less is formed from glycerol - Glycerol kinase Acylation of the two free hydroxy goups Formation of phosphatidic acid Step 1: formation of the fatty acyl ester of glycerol - Phosphatidic acid

Iron-Sulfur proteins

Fe is here not in a heme group but bound to inorganic S or Cys-S Simple to more complex Fe-S centers Always used in 1 e^- transfers with one Fe in cluster At least 8 Fe-S proteins known in mitochondria electron transport Potential vary form -0.65 to + 0.45 V, depending on the microenvironment

Oxidative Phosphorylation - overview

Formation of ATP by oxidation of glucose, fatty acids and aminoacids Oxidative phosphorylation Photophosphorylation

Formation of Malonyl-CoA in fatty acid biosynthesis

Formed from Acetyl-CoA Catalyzed by acetyl-CoA carboxylase Contain a biotin prosthetic group The two-step reaction as similar to other biotin-dependent carboxylation reactions: - Pyruvate carboxylase - Propionyl CoA carboxylase Biotin known as Vitamin H or B7 Intestinal bacteria generally produce an excess of the body's daily requirement - Deficiency is rare

P-class pumps

Found in - The plasma membrane of plants, fungi, bacteria (H+ pump) - Plasma membrane of higher eukaryotes (Na/K+ pump) - Apical plasma membrane of mammalian stomach (H+/K+ pump) - Plasma membrane of all eukaryotic cells (Ca2+) - Sarcoplasmic reticulum membrane in muscle cells (Ca2+ pump)

Recap of Gluconeogenesis

Gluconeogenesis is the reverse glycolysis - Takes place mainly in the liver Build up of glucose to be sent in the blood stream when the blood sugar is low - Fasts, exercise Build up of glucose storage (glycogen) after a meal when blood sugar is high Costs more energy than glycolysis produces - Costs 6 high energy phosphate groups (glycolysis produces 2) - To ensure irreversible steps Glycolysis has 3 irreversible steps that needs to be bypassed in gluconeogenesis (other enzymes are the same)(Selvom det første irreversible step er delt I to, gælder det for ét step) - Step 1, 3 and 10 of the glycolysis Molecules that can enter the gluconeogenesis are called glucogenic - 18 of the 20 common amino acids

V-class proton pumps

Found in: - Vacuolar membranes in plants, yeast, other fungi. - Endosomal and lysosomal membranes in animal cells - Plasma membrane of osteoclasts and some kidney tubule cells.

F-class proton pumps

Found in; - Bacterial plasma membrane. - Inner mitochondrial membrane. - Thylakoid membrane of chloroplast.

ABC superfamily

Found in; - Bacterial plasma membranes (amino acid, sugar, and peptide transporters) - Mammalian plasma membranes (transporters of phospholipids, small lipophilic drugs, cholesterol, other small molecules).

The four pathways of the glyoxylate cycle

Four pathways are involved - Fatty acid breakdown - Glyoxylate cycle -Citric acid cycle - Gluconeogenesis Regulation: - Isocitrate routed to the glyoxylate cycle or CAC - To CAC: Isocitrate dehydrogenase -Activated by low energy signals and CAC intermediates: CAC starts producing NADH/ATP -Inhibited by phosphorylation (PK) - To Glyoxylate: isocitrate lyase -Eventually leads to gluconeogenesis -Deactivated by low energy signals and CAC intermediates

Electron carriers in multienzyme complexes

Four unique electron-carrier complexes Each complex can do its part of pathway by itself - Complex I goes from NADH to Q - Complex II goes from Succinate in TCA to Q - Complex III goes from Q to Cyto c - Complex IV from Cyto c to O2

Conversions of one-carbon units on tetrahydrofolate

From the top grey area: Methyl group (most reduced form) Methylene group (intermediary oxidized) Methenyl- , formyl- or formimino-group (most oxidized)

Gibbs free energy

G is he amount of an energy capable of doing work during a specific reaction - Pressure and temperature has to be constant - The change of energy during the reaction is denoted deltaG; Reactions where free energy is released deltaG is negative and the process is exergonic;When energy is used, deltaG is positive- endergonic G depends on both H and S: deltaG = deltaH - TdeltaS - deltaG will be lowered when heat is released (negative deltaH) or when disorder increases (positive deltaS); Typical for energetically favorable processes (spontaneous) High positive deltaG = not likely to occur - requires much energy High negative deltaG = releases much energy When deltaG is around zero the reaction is near equilibrium no net movement of the reaction and thus no energy capable of doing work The unit of deltaG is J/mol - Gibbs free energy is the kind of energy cells use (cannot use heat for example)

Regulation of glyceroneogenesis

Glucocorticoid hormones - Stimulates glyceroneogenesis and gluconeogenesis in liver - Suppress glyceroneogenesis in adipose tissue Diabetes millitus - High levels of fat in the blood interferes with glucose utilization in muscle and promote insulin resistance Thiazolidinediones - Activate peroxisome proliferator activated receptor γ (PPARγ) - Activation of PEP carboxykinase

Regulation of the ATP producing pathways

Glycolysis - Product inhibition - Regulation by the levels of Pi, AMP, ADP and ATP - The places of regulation Citric acid cycle - Product inhibition - Regulation by the levels of Pi, AMP, ADP and ATP - Regulation by the levels NADH and NAD+ - The places of regulation Oxidative phosphorylation - Electron transport chain

The pentose phosphate pathway

Glucose 6-P can be redirected from the glycolysis, into the pentose phosphate pathway - Glucose 6-phosphate + H2O + 2NADP+ → NADPH + 2H+ + CO2 + Ribulose 5-phosphate - 3 step reaction Ribulose 5-P is then either: - Converted to Ribose 5-phosphate; Precursor of DNA, RNA, Coenzymes - Recycled into glucose 6-phosphate; 6xRibose 5-P makes 5xGlucose6-P; Does not use or produce NADPH; Continuous production of NADPH (and CO2 as waste) The glucose 6-phosphate comes from the start of the glycolysis - It can thus be 'rerouted' to the pentose phosphate pathway - After the third step (irreversible) of glycolysis (Fructose 6-P → Fructose 1,6-BisP) the compound can only go through glycolysis - This step in the glycolysis is therefore called the committed step!

Glycolysis (important facts)

Glucose is degraded into 2 pyruvates Released energy is conserved in the form of ATP and NADH (2 of each) Divided into two phases - Preparatory and payoff phase Consists of 10 steps Other compounds enter the pathway(andre sukre) The end product, pyruvate, is an important metabolite in various pathways Step by step walk-through - Overall change of the compound, the facilitating enzymes, cofactors involved

Glutamate and Glutamine as NH4+ carriers

Glutamate is more abundant than the other amino acids Because of its chemical instability and importance for cell growth and function, it is critical that the delivery of L-glutamine be optimized to each unique cell culture process. Hence the effective use of L-glutamine and L-glutamine equivalents in cell culture requires an understanding of its chemistry and multiple delivery forms. For a more complete discussion of L-glutamine as a cell culture additive go to Sigma's Media Expert.

Proposed mechanism for glutamine amidotransferases

Glutamine is the major source of [N] of most metabolites Enzyme group: glutamine amidotransferases

Glycerol - Oxidation of Fat and fatty acids

Glycerol contribute to only 5% of the energy of triacylglycerols Enter glycolysis as D-glyceraldehyde 3-phosphate.

Two metabolic fates of glycine

Glycine is degraded by three pahways - Glycine is converted to serine by addition of a hydroxymethyl group - Glycine undergoes oxidative cleavage to CO2 and NH4+ and a methylene group - Conversion of glycine to glyoxylate which is further oxidized to oxalate

Recap of the metabolism of glucogen in animals

Glycogen is stored glucose in liver (blood sugar buffer) and muscles (energy supply) Glycogen is long branched sugar chains which are broken down to G1-P by glycogen phosphorylase and synthesized by glycogen synthase The glycogen debranching (or branching) enzyme removes (or creates) glycogen branches - The branches ensure high solubility and many locations for reaction Phosphoglucomutase facilitates the G1-P ↔ G6-P transition (G6-P enters glycolysis/gluconeogenesis) UDP is an important mediator of glycogen synthesis

Glycogen regulation

Glycogen phosphorylase (break down) has two steric forms - Phosphorylase a (highly active) - Phosphorylase b (less active) Down regulation - Liver: Abundant glucose binds to phosphorylase a, inactivating it - Muscle: At rest, the phosphorylase a phosphatase dephosphorylates the phosphorylase a to the b-form Stimuli to the active form: - Liver: Glucagon - Muscle: Epinephrine , AMP and Ca2+ - Activate Phosphorylase b kinase resulting in the active phosphorylase a-form

Recap of Coordinated regulation of glycogen synthesis and breakdown

Glycogen phosphorylase (glycogen break down) has an highly active and a less active form - Liver: Activated by glucagon, inhibited by glucose - Muscle: Activated by epinephrine, Ca2+ and AMP, inhibited by phosphorylase a phosphatase Epinephrine and glucagon initiates and enzyme cascade (amplification of signal) leading to high glycogen → G1-P conversion Glycogen synthase is inhibited by glucagon and epinephrine and stimulated by insulin The metabolic regulation of carbohydrates differs from myocytes to hepatocyes

Glycogen regulation

Glycogen synthase (build up) shows similar regulation as glucogen regulation - Active a-form - Inactive b-form (phosphorylated) The phosporylation is controlled by at least 11 different kinases Insulin induces the dephosphorylation of the inactive form and prevents phosphorylation of the active form - Reciprocal regulation Glucagon and epinephrine inhibits formation of the active form - Prevents glycogen synthesis

Formation of glycogen granules

Glycogenin primes the initial sugar residues - Sugar residues comes from UDP-sugar - 8 Residues are put together in the primer before glycogen synthase starts its polymerization - In the beginning the glycogen is heavily branched - Glycogenin will keep sticking on the reducing end

Route of Glucose 6-phosphate (pp pathway or Glycolysis)

Glycolysis and Pentose phosphate pathway share metabolites Glycolysis: - ATP and NADH - Main pathway PP pathway: - NADPH Regulated by the NADPH level - Glucose 6-P dehydrogenase; Entry to PP pathway

Recap of Glycolysis

Glycolysis is divided in a preparatory and a payoff phase The overall reaction of glycolysis: - Glucose + 2NAD+ +2ADP + 2Pi → 2pyruvate + 2NADH + 2H+ +2ATP + 2H2O Separate endergonic and exergonic processes: Glucose + 2NAD+ → 2pyruvate + 2NADH + 2H+ ΔG'^o = - 146 kJ/mol 2ADP + 2Pi → 2ATP + 2H2O ΔG'^o = 61 kJ/mol Net: ΔG'^o = -85 kJ/mol Glycolysis releases only a minor part of glucose's energy - Pyruvate has much chemical energy - Citric acid cycle and the respiratory chain (to come) The glycolysis is tightly regulated to ensure a constant amount of ATP in the cells All 9 intermediates are phosphorylated - Hydrophillic and cannot pass cell membrane (godt så de ikke smutter ud inden de er omdannet) Glycolysis has three irreversible steps

Dehydration step 3 in fatty acid synthesis

H2O is removed from C2 and C3 trans-Δ2-butenoyl-ACP is formed The reaction is catalyzed by β-hydroxyacyl-ACP dehydratase (DH)

The enzyme complex citric acid cycle

Has five coenzymes or prosthetic groups - Bound (recycled): Lipoate, TPP and FAD - Free: CoA (to acetyl-CoA), NAD+ (to NADH) Has multiple copies of three different enzymes E1: Pyruvate dehydrogenase (yellow) - TPP is bound in the active site E2: Dihydrolipoyl transacetylase (green) - has three functionally distinct domains E3: Dihydrolipoyl dehydrogenase (red) - FAD is bound in the active site

Enthalpy

Heat content of a reacting system - Release heat (exothermic reaction) - Take up heat (endothermic reaction) - From the systems point of view (not the surroundings) Dependent on the number and type of bonds broken or created during a reaction deltaH is the change in enthalpy of reactants - deltaH is negative for exothermic and positive for endothermic reactions

Cytochromes

Heme type iron containing proteins Have strong absorbance in visible range Classify in to three main groups, a, b, and c: - a's absorb about 600 nm (lowest E) - Heme has a long hydrophobic tail - b's absorb about 560 nm - Standard heme - c's absorb about 550 (highest E) -Covalently attached heme There are subtypes - b562 is a b cytochrome with an absorbance specifically at 562nm Hemes of a and b type closely associated to protein - not covalently bound to protein Heme c's are covalently bound to protein through cys linkages Most cytochromes are integral membrane proteins One exception - Mitochondrial cytochrome c is peripheral protein bound to outer surface of inner mitochondrial membrane Standard reduction potential of the heme iron depends on its interaction with the protein

Prompt energy and storage

High energy demanding tissues (like muscle) have phosphocreatine, PCr - High energy phosphate group - PCr + ADP <-> ATP + Cr deltaG'^o = -12.5 kJ/mol - Fast buildup of ATP levels during sudden energy demands - Catalyzed by creatine kinase (cofactor Mg2+) - PCr restored at rest All cells have chains of Pi - Also an energy reservoir to restore ATP levels

Genetic defects in the urea cycle

Humans with a defect in one of the urea cycle enzymes cannot tolerate protein-rich diets This can result in hyper-ammonemia or in the built-up of one of the intermediates Treatment with benzoate or phenylacetate can lower the levels of ammonia in the blood stream The ammonia is introduced via glycine and glutamine

Inhibitory protein prevents ATP hydrolysis during Hypoxia (Ischemia)

Hypoxic (ischemic) cell is deprived of O2 - Can happen during heart attack or stroke If no O2 is present electron transfer to O2 stops and then the proton pumping ceases The proton-motive forces colaps - Under such conditions ATP synthase could start to run in reverse - Degrade ATP to ADP to pump protons out of cell to keep the gradient This is prevented by protein inhibitor IF1 - 84 residue protein - Bind to 2 ATP synthase molecules and inhibits activity - Only works when in dimer form - Only works when in dimer form, which is the case at pH < 6.5 - pH only <6.5 if pyruvic or lactic acid have built up because of O2 debt

The citric acid cycle

In CAC, acetyl-CoA is oxidized to CO2 to maximize the harnessed energy - Direct oxidation would give CO2 and CH4: No energy will be obtained The path to the oxidation is critical - Steps are either; 1. Energy conserving oxidation (ATP/NADH) 2. Organizing functional groups for oxidation; Carbonyl groups are important for facilitating reactions The citric acid cycle contains eight steps - Cycle: end product oxaloacetate is used in the first step - Step by step (look at picture)

The Citric Acid Cycle Step 7

In a two-step reaction, fumarase converts fumarate to L-malate - OH- is added to C-3 reducing a C-C double bond to single bond - Carbanion transition state - An H+ is added to C-2 - L-stereoform of malate - Reversible

The three groups of C-C bond formation/break

In aldol and claisen ester condensation a Nucleophile carbanion reacts with an electrophile carbonyl. In the decarboxylation of a beta-keto acid CO2 leaves assisted by the carbonyl. Homolytic cleavage.

Ketone Bodies Used as Fuels

In extrahepatic tissues 1. D-beta-hydroxybutyrate is oxidized to acetoacetate - Catalyzed by D-beta-hydroxybutyrate dehydrogenase 2. Formation of the CoA ester - Transfer of CoA from succinyl- CoA - Catalyzed by beta-ketoacyl-CoA transferase 3. Cleavage of the acetoacetyl-CoA to two acetyl-CoAs - Catalyzed by the enzyme thiolase - Acetyl-CoA enter citric acid cycle

Desaturation of fatty acids in fatty acid biosynthesis

Introduction of double bonds - Palmitoleate 16:1 (Δ9) and oleate 18:1 (Δ9) - Fatty acyl-CoA desaturase - A mixed function oxidase - In the endoplasmatic reticulum - Mammalian hepatocytes can only introduce a double bond at Δ9

Other complexes, than the multienzyme complexes, that pass electrons to Ubiquinone

In fatty acid oxidation first step catalyzed by acetyl-CoA dehydrogenase - Take 2E to oxidize a fatty acid and put on FAD of enzyme - Transfer e- to electron transferring flavoprotein (ETF) - ETF passes to ETF:uniquinone oxidoreductase Glycerol-3-P in cytosol - Comes from glycerol of triacylglycerols - Of reduction if dihydroxyacetone phosphate in glycolysis Enzyme: glycerol-3-P dehydrogenase - On outer face of inner mitochondria membrane - Transfers electrons to Ubiquinone Used to shuttle reducing equivalent between - NADPH in cytosol and NAD in mitochondria

Human Digestive Tract

In humans, most of the degradation of ingested proteins take place in the gastro-intestinal tract Secretion of gastrin is stimulated, which further stimulate the secretion of HCl and pepsin Pepsin is secreted as pepsinogen, which is activated to pepsin by autocatalysis Hydrolyze the bond on the amino-terminal side of Phe, Trp and Tyr In small intestine, the low pH trigger secretion of secretin into the blood Secretin stimulate pancreas to secrete bicarbonate, which neutralize HCl and increase the pH to about 7 Amino acids in the upper part of small intestine cause the release of the hormone cholestokinin to the blood Cholestokinin stimulate secretion of pancreatic enzymes Pancreatic enzymes: - trypsinogen - chymotrypsinogen - procarboxypeptidases A and B The active enzymes have optimal activity around pH 7 and 8 The free amino acids enter the blood capillaries in the villi and travel to the liver

Hypoxia lead to ROS production and several adaptive responses

In hypoxia: - imbalance between input electrons into the chain and O2 to finish Under these conditions start to build up ROS Removal of ROS - SOD, GSH-Red Two additional control mechanisms 1. Slow down Pyruvate dehydrogenase (PDH) - Phosphorylated by a PDH kinase to inactivate PDH 2. Swap out a subunit of Complex IV that is optimized for normal - O2 levels (COX4-1) - with a subunit better suited for hypoxic conditions (COX4-2) Both are controlled by Hypoxia Inducible Factor (HIF-1)

Alternative pathways for phenylalanine

In individuals with PKU, phenylalanine is converted to phenylpyruvate These are excreted in the urine giving the urine a characteristic odor

Glycogen

In invertebrates, excess glucose is stored as glycogen, mainly in the liver and in the muscles Glycogen is long chains of glucose residues - Energy storage - Prevents osmotic disturbance - 0.01 µM glycogen (liver) would be 400 µM as free glucose The muscle glycogen is used to provide a quick source of energy - Strenuous exercise - 1 h reserve Liver glycogen functions as a glucose buffer - Ensures a stable blood sugar between meals - 12-24 h reserve The formation of glycogen is called glycogenesis The break down is called glycogenolysis - Gut: Enabling transport of glucose into the cells - Muscle: For glycolysis to obtain energy - Liver: To produce glucose to be secreted into the blood Three enzymes are involved in glycogenolysis in muscle/liver - Glycogen phosphorylase - Glycogen debranching enzyme - Phosphoglucomutase

Electron carriers

In many enzyme-catalyzed oxidations, electrons are transferred to electron carriers - NAD, NADP, FAD and FMN (+ more) NAD and NADP strips off 2 protons and 2 electrons - Both electrons and one proton reacts with NAD(P) to give NAD(P)H - The last proton is released to the solution

Fates of glucose 6-P

In muscles the glucose 6-P enters the glycolysis to provide energy In the liver it is transformed into glucose by glucose 6-phosphatase (only found in the liver - gluconeogenesis) and released into the blood For separation from glycolysis the reaction takes place in the ER

Catabolic pathways for phenylalanine and tyrosine 1

In the breakdown process of phenylalanine, several inheritable genetic defects have been observed - Specific intermediates accumulates, causing defective neural development - Defect of phenylalanine hydroxylase is responsible for the disease phenylketonuria (PKU) - Phenylalanine hydroxylase belongs to the group of enzymes named mixed-function oxidases

Recap of the Pentose phophate pathway of Glucose oxidation

In the pentose phosphate pathway, glucose 6-P is converted into ribulose 5-P and NADPH Ribulose 5-P can have two purposes - Conversion to ribose 5-P and eventually DNA, RNA or coenzymes - Recycling to glucose 6-P in a non-oxidative pathway for continuous production of NADPH If glucose 6-P is converted to fructose 6-P instead and then further to fructose 1,6-BisP through an irreversible step, it has to go through the glycolysis (called the committed step) Transketolase and transaldolase convert 6 isoforms of ribulose 5-P into 5 glucose 6-P by various carbon-group transfers The NADPH level regulates whether glucose 6-P enters the glycolysis or the pentose phosphate pathway by inhibiting glucose 6-phosphate dehydrogenase

ABC transporter mechanism

In the resting state, ATP hydrolysis is prevented because these motifs are held apart (A). The binding protein, BtuF, performs a dual role: It delivers B12 to the mouth of the transporter as it binds tightly to stabilize the transporter in the catalytic transition state conformation that promotes ATP hydrolysis (B). In this conformation, portions of the two BtuD subunits have moved together to complete the ATP-binding sites, BtuF has relinquished its grip on B12, and rearrangements in the transmembrane helices have opened the translocation pathway from the binding protein to the cytoplasm. A vestibule located in the cytoplasm between the four subunits permits B12 to exit even while the BtuD subunits are still tightly engaged. After ATP hydrolysis and B12 release, the transporter returns to the original state and BtuF is released (C).

Ketone Body Formation and Export from the Liver

Increased gluconeogenesis (un-treated diabetes, low-calory intake) - Depletion of citric acid cycle intermediates - Fatty acids enter mitochondria and degraded to acetyl-CoA - Acceleration of the formation of ketone bodies - Increased levels of blod levels of acetoacetate and -D-hydroxy-butyrate lower the pH of the blood (acidosis) - High levels of ketone bodies is observed in blood and urine (ketosis)

Regulation of the level of NH4+

Indicate possible ways of regulation of the level of NH4+: Oxidative deamination - Regulate the formation of NH4+ - GTP enhance / ADP reduce Formation of carabmoyl phosphate - Regulate the removal of NH4+ - N-acetylglutamate enhance formation (Acetyl-CoA, glutamate and arginine enhance)

Stored Fat - inflammation

Influence of minor dietary lipid components carried by chylomicron remnants on the release of pro-inflammatory molecules and NO by foam cells in the subendothelial space. CM, chylomicron; CMR, CM remnant; ICAM-1, intracellular adhesion molecule-1; IL-1β, interleukin 1β; TNFα, tumour necrosis factor α; - inhibition; + stimulation.

Effect of Aspirin

Inhibits the COX enzyme Reduce fever and pain Acetylation of Ser in the active site of COX enzyme Nonsteroidal anti-inflammatory drugs (NSAID) Specific COX-2 inhibitors - Celecoxib - Rofecoxib

Regulation of the formation of triacyl glycerols

Insulin stimulate the conversion of carbohydrates and amino acids to fat In untreated diabetes militus this conversion is also impaired - Acetyl-CoA is instead converted into keton bodies

Receptor with no intrinsic enzyme activity

Interacts with cytosolic protein kinase, which activates a gene-regulating protein (directly or through a cascade of protein kinases), changing gene expression.

Maintaining high driving forces by maintaining products

Keeping deltaG at a high negative value can be obtained by a change in the product 'D' is kept low in the equation deltaG is kept highly negative

Citric acid cycle - Coenzyme A

Key part is the reactive thiol group - Acyl binds here, creating a thioester - High transfer potential (-31.5 kJ/mol)

Pyruvate → Lactate

Lactic acid fermentation by lactate dehydrogenase Anaerobic metabolism Heavy muscle activity No oxygen is present - pyruvate cannot be oxidized - Instead it is reduced to lactate - Production of NAD+ - No net change in NAD+ and NADH when including glycolysis (we use NADH for energy production) - Helps drive the glycolysis, but not a further energy yield

Branched - chain amino acids

Leucine, isoleucine and valine are oxidized as fuel in extrahepatic tissue - Muscle - Adipose - Kidney - Brain Branched-chain aminotransferase is not found in the liver - Formation of the corresponding α-keto acids Branched-chain α-keto acid dehydrogenase complex catalyze an oxidative decarboxylation - Form an acyl-CoA product Analogous to complexes catalyzing - Oxidation of pyruvate to acetyl-CoA - Oxidation of α-ketogluturate to succinyl-CoA

Oxidation of polyunsaturated fatty acid

Linoleate (cis-delta9,cis-delta12, C18): - Three passes through the standard b-oxidation sequence --> cis-delta^3,cis-delta^6-docenoyl-CoA Enoyl-CoA isomerase --> trans-D2,cis-D6-docenoyl-CoA --> one cycle b-oxidation --> trans-D2,cis-D4-C10-CoA 2,4-dienoyl-CoA reductase --> trans-D3-C10-CoA Enoyl-CoA isomerase --> trans-D2-C10-CoA

Gluconeogenesis - reverse glycolysis

Mainly in the liver Build up of glucose for - Maintenance of blood sugar; Drops at fasts and exercise; Important for the brain - Energy storage; After a meal (high bloodsugar); Monosaccharides -> Glycogen, starch - Other monosaccharides and disaccharides; Costs energy

The Citric Acid Cycle Step 8

Malate dehydrogenase converts the L-malate into oxaloacetate - Enters the cycle again (acetyl-CoA) - NADH is produced - Low oxaloacetate concentration ensure a low ∆G - Reversible

Pathways to phosphatidylcholine and - ethanolamine

Mammals used "strategy 2" - Combining phosphorylated and activated head group with - Diacylglycerol Phosphatidylserine: - Head-group exchange In the liver: Phosphatidylcholine is formed from phosphatidyl-ethanolamine by methylation PSS: Phosphatidylserine synthase

Glycolysis feeder pathways

Many other carbohydrates enter the glycolysis - Glucogen and starch (storage or diet) - Poly- Di- and monosaccharides from diet (Poly and di are converted to mono in intestine, taken up and transported to the liver) Storage glycogen is degraded into monosaccharides - Glucose 1-phosphate by glycogen phosphorylase and Pi - ATP is not used for this reaction Glucose 1-P is transformed into glucose 6-P - Phosphoglucomutase - Enters the glycolysis Phosphorylated carbohydrates cannot pass the membranes - The carbohydrates are hydrolyzed rather than phosphorylyzed in the intestine before being transported into the cells and further into the blood stream - Dietary glycogen is hydrolyzed by amylase to glucose (no P)

Protein phosphorolylation and dephosphorolation

Many signal pathways Many signal pathways include phosphorylation cascades In this process A series of protein kinases add a phosphate to the next one in line, activating it. Phosphatase enzymes then remove the phosphates.

Enzyme regulation

Metabolites are shifted between pathways depending on the shifting needs of the cell - Carbohydrates, lipids, amino acids, nucleosides The reaction rates/directions are generally regulated on enzyme level - Change of number of enzymes catalyzing the reaction - Change in the catalytic activity of the enzymes (by inhibitors/activators) The regulating signals can come from metabolites, ions, hormones, cytokines etc. The speed of the regulation matches the speed of the changes - Bone marrow stem cells differentiate to erythrocytes (slow) - Stimulation of glycogen synthesis (response to meal, medium) - Stimulation of glycogen phosphorolysis (low blood sugar, fast) Regulating mechanisms can be divided in 10 steps

Mitochondria - functions

Mitochondria have different forms Cells with high demand of ATP do contain many mitochondria per cell - These mitochondria have more, and more condensed packed cristae Oxidative phosphorylation Other functions - Thermogenesis - Steroid synthesis - Apoptosis - Store of Ca2+ Mitochondrial misfunction is related to - Neurodegenerative diseases - Cancer - Diabetes - Obesity - Ageing (?)

Mitochondrial genes

Mitochondria have their own double stranded circular DNA and their own ribosomes - In total 37 genes (16,569 bp) - Only a fraction of the proteins found in mitochondria - Mostly the membrane proteins and a few rRNA and tRNA Bulk of proteins, about 900, are made in cell nucleus synthesized on cytosolic ribosomes and then imported to mitochondria after synthesized

Mitochondria - struture

Mitochondria have two membranes This gives four compartments: 1. Mitochondrial matrix 2. Inner membrane 3. Outer membrane 4. The intermembrane space

Role of tetrahydrobioterin

Mixed-function oxidase: simultaneous hydroxylation of a substrate by an oxygen atom and reduction of the other oxygen atom to H2O Tetrahydrobiopterin is the cofactor which carries electrons from NADH to O2 It is subsequently reduced by the enzyme dihydropterin reductase

Mixed Function Oxidases - 3

Monooxygenase - Only one oxygen atom is incorporated in the substrate - The other one is reduced to water - The general reaction equation include two substrates, A and B: AH + BH2 + O2 --> A-OH + B + H2O Other names: - Hydroxylases - Mixed-function oxidases - Mixed-function oxygenases

Amino group catabolism in vertebrate liver

Most amino acids are metabolized in the liver Amino acids are recycled and reused Glutamate and glutamine play critical roles in nitrogen metabolism -In hepatocytes, the amino group is transferred to alpha-ketoglutarate forming glutamate - In extahepatic tissue, excess of NH4+ is converted to glutamine - In skeletal muscles, amino groups are transferred to pyruvate to form alanine - Oxidative deamination take place in the mitochondria of hepatocytes

Mitochondrial P-450 oxygenase catalyzes steroid synthesis

Most steroid hormones are synthesized by hydroxylating of cholesterol These reactions are done by a family of heme-containing enzymes called Cytochrome P-450s because they absorb light at 450 nm. These enzymes are located on inner membrane of mitochondria They catalyse a one-atom of molecular oxygen transfer to the substrate: R-H + O2 + NADPH + H+ → R-OH + NADP+ + H2O Steroidogenic cells contain a lot of mitchondria specialized for steroid synthesis Other cytochrome P-450 families - In endoplasmatic reticulum of hepatocytes, and in epithelial cells - Substrates: Hydrophbic compounds, including xenobiotics - Broad and overlapping substrate specificities - The aim is to make the hyrophobic compounds more water soluble, for excreation

Fatty acid synthase - fatty acid biotransformation

Multienzyme complex - Seven separate polypeptides (core complex) - Additional three polypeptides are involved The intermediates of the reaction are covalently bound to the complex - Two thiol groups of the synthase complex (β-ketoacyl-ACP synthase and Acyl carrier protein)

Chemiosmotic model

Net reaction will be ADP + Pi + nH+P → ATP + nH+N

Nitrogenase complex

N2 is fixed through the Nitrogenase complex

NAD(H) in reaction assays

NAD and NADH have different absorption patterns - Used by biochemists to assay reactions involving NAD-dependent dehydrogenases

Summary of the flow of electrons and protons in oxidative phosphorylation

NADH + H+: 4 H+ + 4 H+ + 2 H+ FADH2: 4 H+ + 2 H+ Overall reaction is efficient Net pathway for transfer of 2 electrons from 1 NADH: NADH + H+ + ½ O2 → NAD+ + H2O Looking at the energy releasing half of the reaction This reaction has a ΔE'O of 1.14 V Using ΔG= -nFE, ΔG'O = -220 kJ/mol - Actually E much higher due to actual concentrations With succinate one get ΔG'O = -150 kJ/mol again much higher in cell For each pair of electrons transferred to O2 - Vectorial equation: NADH + 11H+N + ½ O2 → NAD+ + 10H+P + H2O

Actors in Amino acid biosynthesis

NH2- from glutamine - glutamine amidotransferases Transamination - PLP Transfer of C1-groups: THFA and S-adenosyl-methionin(AdoMet) PRPP - C atoms in His and Trp NAD(P)H - reducing power HS-CoA - transfer acetyl and succinyl groups PAPS (3'-Adenosine 5'-phospho-sulfate) (S in Cys and Met)

Transfer of acetyl groups to cytosol - as citrate

Nearly all Acetyl-CoA is formed in mitochondria - Pyruvate oxidation - Catabolism of the carbon skeletons of amino acids

Mobilization of stored triacylglycerols

Need for metabolic energy: 1. Hormone signaling to adipocyte - bind its receptor 2. Stimulate adelyate cyclase, producing cAMP, which activate PKA 3. PKA activate the hormone sensitive lipase 4. Phosphorylate perilipin 5. Hydrolysis of triacyl glycerol 6. Fatty acid leaves adipocytes 7. Moved to tissue (skeletal muscle, heart and renal cortex) 8. Oxidation of fatty acids

Electron carriers - in oxidative phosphorylation

Nicotinamide nucleotide Flavin nucleotides

Citric acid cycle - Fates of pyruvate

No oxygen: - Lactate: Hard working muscle - Ethanol: Plants, microorganisms - Fermentation - Keeps glycolysis running Oxygen: - Main pathway - Acetyl-CoA - Citric acid cycle (CAC) - Oxidized to CO2 - NADH and FADH2: ATP in respiratory chain (O2) - Called cellular respiration

Other roles of the Citric Acid Cycle

Not confined to oxidation of acetate Many intermediates leave the cycle as precursors - anabolic reactions (or enters - catabolic) - Fatty acids - Most amino acids - Pyrimidines/Purines: Nucleic acids Both catabolism and anabolism - amphibolic Metabolites are reused in the cycle Removed precursors has to be replenished - Anaplerotic reactions (red arrows) - Pyruvate carboxylase - Most important - Stimulated by Acetyl-CoA

Shuttle systems are required to oxidize cytosolic NADH in mitochondria

Not only does ATP need to get transported, but the NADH generated in glycolysis also must be transported to mitochondria so it can get oxidized and regenerated NAD+ Malate-aspartate shuttle is used in liver kidney and heart - Net effect: NADHP → NADHN Glycerol 3-phosphate shuttle is used in skeletal muscles and brain: - Net effect: NADHP → FADH (mitochondrial membrane) Step 1 (cytosol) oxaloacetate is reduced by cytosolic NADH to make malate and regenerates NAD+ Malate dehydrogenase - Note: this is the REVERSE of the reaction that normally occurs in the mitochondria Step 2 In an antiport system malate goes into mitochondria and α-ketoglutare comes out Malate α-ketoglutarate transporter Step 3 In normal TCA reaction malate is oxidized to α-ketoglutarate to generate NADH, - malate dehydrogenase - thus the cytosolic NADH has appeared in the mitochondria - But this have also increased the amount of oxaloacetate in the TCA cycle, - throw off the balance Step 4 Oxaloacetate is transaminated to aspartate - Glutamate is the NH2 donor - Aspartate amino-transferase - This change glutamate into an α-ketoglutarte that may be used in the in the malate/α-ketogluatate transporter Step 5 Aspartate is transported out and glutamate transported in using - glutamate aspartate transporter - so that is where the Glu came from! Step 6 Aspartate is deaminated and converted back to oxaloacetate - Aspartate amino tranferase - Now in the cytosol - NH2 is transferred to α-ketogluarate to make glutamate

Recap of Bioenergetics and Biochemical reaction types

Nutrients are used for building blocks, precursors and energy - The breaking down of nutrients is called catabolism (deltaS is positive, deltaG is negative, energy yielding process) - The building up of cellular components is called anabolism and costs energy Bioenergetics is quantification of energy - The energy connected to the biological processes is called Gibbs free energy (G) and obey the laws of thermodynamics - The change of free energy, deltaG, at each reaction can be calculated from the enthalpy (H) and entropy (S): deltaG = deltaH - TdeltaS - For standardizing values we introduce the term deltaG'^o which is the change of free energy at 25C, 1 ATM pressure, 55.5 M water, 1 mM Mg2+ and pH 7 - The actual deltaG, however, can be far from the standard value The operational sign of deltaG determines whether the reaction is spontaneous or not - Negative deltaG means a thermodynamically favorable reaction and is thus spontaneous - The reaction thus strongly depends on the concentration of the reactants and products High activation energies prevent or slow down reactions - Enzymes decreases the activation energy and speed up the reaction rate; This does not change the equilibrium constant nor the free energy of the reaction

Oxidative phosphorylation is regulated by cellular needs

O2 consumption tightly regulated, limited by availability of ADP and Pi - named acceptor control Acceptor control ratio can increase over 10 fold over basal rate, when ADP is around In general cell maintains high [ATP] / [ADP][Pi] ratio Starting energy consuming processes will increases the amount of ADP - then respiration takes off to increase the ATP production Overall control is so good, that very little change in [ATP], [ADP], or [Pi] is ever observed ATP is formed only as fast as it is used in energy-requiring cellular activities

Nitrogen-fixing nodules in legumes

O2 destroys Nitrogenase, reductase half-life 30 sec Dinitrogenase half-life 10 min Leg-hemoglobin binds O2 Plant produces the apo-protein Bacteria produces heme

Non-oxidative recycling phase (pentose phosphate pathway)

Occur in tissue that needs NADPH Ribulose 5-P is epimerized to Xylulose 5-P - Epimerase: an isomerase interconverts epimers Then an interplay between transketolase and transaldolase transfers carbon groups between Xylulose 5-P, Ribose 5-P and their intermediates

Regulatory step of pathway

Often, the first and last step of a pathway has high negative ∆G and are the regulated steps (or at branching points) Example in figure: All three steps have a conversion rate of 10 (molecules/time) ∆G is near 0 in step 2 and 3 - Forward rate is almost the same as reverse rate - Can easily be turned in the reverse reaction ∆G for reaction 1 is large and negative - Irreversible (has to be bypassed) - Often the end product (here 'D') is an allosteric inhibitor of reaction 1 - 'Shuts down' the pathway when enough product has been made.

Proton-Motive Force also used for active transport

One last puzzle piece: - If you make ATP in the mitochondria how do you get it into the cytosol, and vice versa with ADP and Pi? Adenine nucleotide translocase exchanges ATP4- on inside with ADP3- on outside - Integral membrane proteins In this process - inside loses a negative charge and - outside gains a negative charge so net is moving a negative out Outside is already + (excess H+) so this is favored by gradient and nothing special has to be done Can be inhibited by atractyloside (a toxic glycoside)

Important cofactors in one-carbon transfer reactions

One-carbon transfer Involve biotin, tetrahydrofolate or S-adenosylmethionine Biotin transfer one-carbon in its most oxidized form (CO2) Tetrahydrofolate transfer one-carbon in intermediate oxidative status S-adenosylmethionine transfers methyl groups, the most reduced state of carbon

Proline can also be made from ornithine in mammalsOrnithine δ-aminotransferase reaction: a step in the mammalian pathway to proline.

Ornithine δ-aminotransferase reaction: a step in the mammalian pathway to proline. This enzyme is found in the mitochondrial matrix of most tissues. Although the equilibrium favors P5C formation, the reverse reaction is the only mammalian pathway for synthesis of ornithine (and thus arginine) when arginine levels are insufficient for protein synthesis.

Membrane transport

Passive Active Carrier proteins Nutrient-uptake Channel proteins Cell potential (uddyb)

Mixed Function Oxidases - 1

Oxidases - Oxidation where oxygen is the electron acceptor - Oxygen do not take directly part of the reaction generating the electron 2H+ + 2e- + O2 --> H2O2 2H+ + ½ O2 + e- --> H2O

Activation of fatty acids

Oxidation of fatty acids in animal cells take place in mitochondrial matrix. Free fatty acids cannot enter mitochondria directly Three enzymatic reactions which activate the fatty acids and transport it to mitochondria matrix: 1. Forming fatty acyl-CoA (Acyl CoA synthase) Fatty acid + CoA + ATP → fatty acyl-CoA + AMP + PPi 2. Formation of fatty acyl-carnitine (Carnitine acyl transferase I) Fatty acyl-CoA + carnitine → fatty acyl-carnitine - Facilitated diffusion through acyl-carnitine/carnitine transporter 3. Forming fatty acyl-CoA (carnitine acyltransferase II) Fatty acyl-carnitine → Fatty acyl-CoA + carnitine (in mitochondria)

Oxidation-reductions (metabolism)

Oxidation: Loss of electrons - Addition of oxygen - Loss of hydrogen Reductions: Vice versa Carbon has different oxidation states depending on its neighbors The release of electrons during oxidation is important for obtaining energy. - The driving force for ATP synthesis (to come)

Flavoproteins - in oxidative phosphorylation

Oxidized flavin nucleotide can accept - One electron (forming the corresponding semiquinone) - Two electrons (forming FADH2 og FMN2) The standard reduction potential of flavin nucleotide depends on the proteins with which it is associated - Functional groups in the protein distort the electron orbitals in the flavin ring, changing the relative stabilities of oxidized and reduced forms. - The relevant standard reduction potential is therefore that of the particular flavoprotein. - The Flavin nucleotide should be considered part of the flavoprotein's active site rather than a reactant or product in the electron transfer reaction. Flavoproteins can serve as intermediates between two-electron donations (as in dehydrogenations) and one-electron acceptance (reduction of a quinone to a hydroquinone).

Mixed Function Oxidases - 2

Oxygenase - Oxygen is incorporated in the substrate Dioxygenase - Both oxygen atoms are incorporated in the substrate

ATP yield from Complete Oxidation of Glucose

Palmitate (16:0 fatty acid) generate 106 ATP via ETC Regulation of the ATP production must follow the cells need

Synthesis of other fatty acids in fatty acid biosynthesis

Palmitate may be elongated - to stearate - or longer saturated fatty acids Fatty acid elongation system - CoA but not ACP is the acyl carrier - Different enzyme systems In smooth endoplasmatic reticulum / mitochondria Linolate and α-linolenate need to be given by the diet

Enzymes in Regulation of glycolysis/gluconeogenesis

Pathway flows are also regulated by changing the number of enzymes - Slow reaction time Regulation of enzyme synthesis is often regulated by hormones Insulin regulates a wide range of enzymes facilitating glycolysis and fatty acid synthesis - Activates transcription factors There are four main transcription factors involved in the regulation of glycolysis/gluconeogenesis - ChREBP and SREBP (stimulated by Xylulose 5-P and insulin, respectively) are increased at high energy levels and facilitates storage of surplus energy as fat (among other things) - CREB (stimulation by glucagon) and FOXO1 increases synthesis of various enzymes facilitating glyconeogenesis As glucagon is a response to low blood sugar, and insulin is a response to high blood sugar, they often regulate the same enzymes or mediators just with the opposite signal - Insulin induces glycolysis and inhibits gluconeogenesis - Glucagon induces gluconeogenesis and inhibits glycolysis - Reciprocally regulation

Regulation of glycolysis/gluconeogenesis (Pathways)

Pathways can go in both directions - Always controlled - depending on the need - Never both ways at the same time - Reciprocally regulated -When one direction is up regulated, the opposite direction is down regulated In glycolysis/gluconeogenesis the regulation is mainly on the conversion controlled by PFK-1 (glycolysis) or FBPase-1 (neo) - Committing step of glycolysis Low energy signals stimulate PFK-1 along with F2,6-BisP (allosteric)

Glycolysis step 6: Glyceraldehyde 3-P → 1,3-Bisphosphoglycerate

Payoff phase starts Inorganic phosphate is added by glyceraldehyde 3-P dehydrogenase - Dehydrogenase: Oxidizes by transferring 1 or more H- to an electron acceptor (usually NAD+) NAD+ is converted to NADH + H+ (two of each now) Reversible

Mixed Function Oxidases - 4

Phenylalanine hydroxylase Fatty acyl-CoA desaturase 1-alkyl-2-acylglycerophospho ethanolamine desaturase

Phosphatidic acid in lipid biosynthesis

Phosphatidic acids is then converted to - Triacylglycerol or - Glycerophosholipid 1,2-diacylglycerol is an intermediate - Phosphatidic acid phosphatase Diacylglycerol is converted to triacylglycerol - Acyl transferase - Fatty acyl-CoA

Glycolysis step 7: 1,3-Bisphosphoglycerate → 3-Phosphoglycerate

Phosphoglycerate kinase transfers the phosphoryl group to ADP in the presence of Mg2+ - Name describes the reverse reaction Energy release is conserved in ATP Reversible - High ATP levels, low ADP levels

Glycolysis step 8: 3-Phosphoglycerate → 2-Phosphoglycerate

Phosphoglycerate mutase relocates the phosphoryl group in a reversible reaction (presence of Mg2+) - Mutase: Subclass of isomerase. Shifts position of a functional group Actually a two-step reaction where phosphoryl is added to C-2 where after the C-3 is removed Reversible

Nucleophilic attacks on ATP

Phosphoryl (PO32-) and not Phosphate (OPO32-) is transferred - P is always transferred to a negative -O of a compound - Acts as an electrophile

Glycolysis step 3: Fructose 6-P -> Fructose 1,6-bisP

Phosphorylation of C-1 by PFK-1 and Mg2+ Second irreversible step of glycolysis - Committing step of glycolysis

Glycolysis step 1: Glucose -> Glucose 6-P

Phosphorylation of C-6 by hexokinase and Mg2+ - Kinase: Transfers phosphate group from a high-energy donor - Phosphorylase: Adds inorganic phosphate to an acceptor Large negative deltaG'° (and deltaG) - is irreversible

Molecules possible for diffusion accross lipid bilayers

Polar and charged substances do not diffuse across lipid bilayers. One way for these important raw materials to enter cells is through the process of facilitated diffusion. Facilitated diffusion depends on two type of membrane proteins: channel proteins and carrier proteins.

Tryptophan as a precursor

Portions of tryptophan yield acetyl-CoA - via pyruvate (blue) - via acetoacetyl-CoA (red) Tryptophan is a precursor for the biosynthesis of other biomolecules

Glycolysis prep. phase

Prep. phase costs two ATP - Priming reactions The compound is cleaved into two trioses.. - Made identical - Glyceraldehyde 3-Phosphate - This yields the payoff fase.. Payoff phase begins - Yields four ATP - Yields two NADH - Product is 2 pyruvates Overall reaction: Glucose + 2Pi + 2ADP + 2NAD+ <-> 2Pyruvate + 2ATP + 2NADH + 2H+ + 2H2O

Glycogen phosphorylase in amino acid oxidation

Pyridoxal phosphate is an essentiel cofactor in the reaction The phosphat group of PLP - General acid catalyst - Promoting attack by Pi on the glycosidic bond

Pyruvates role in Regulation of glycolysis/gluconeogenesis

Pyruvate can go into several pathways - Key metabolite - also regulated Main pathway is citric acid cycle - Acetyl CoA is the first step If no energy is needed acetyl-CoA concentration will build up - Acetyl-CoA inhibits pyruvate dehydrogenase and stimulates pyruvate carboxylase (gluconeogenesis)

Gluconeogenesis bypassing step 10 of glycolysis

Pyruvate → PEP - Divided in a two step reaction First step: Pyruvate (+ HCO3-) → Oxaloacetate pyruvate carboxylase - Second step: Oxaloacetate → PEP + CO2; PEP carboxykinase (name for reverse reaction). Before oxaloacetate can exit the mitochondria it has to be converted to malate - Re-converted in the cytosol -Contributes to NADH concentration in the cytosol Pyruvate from lactate follows a different pathway - NADH is still produced in cytosol Irreversible reaction - Costs a lot of energy (1 ATP and 1 GTP) - In the other direction 1 ATP is produced

Mixed Function Oxidases - cosubstrates

Reduced flavin nucleotides - FMNH2 or FADH2 Reduced adenin dinucelotides - NADH or NADPH α-ketoglutarate Tetrahydrobioterin

Measuring reduction potentials

Quantification of the force that drives the electron transfer between two species - Movement of electrons - Reduction potential = E Standard conditions (reference point) - 1 M H+, 25°C, 1 ATM H2 gas - Standard reduction potential E° = 0 V - Biochemists use pH 7 (E'°) Connected with test cell - 1 M of species A - Depending on the reduction potential of species A, the electrons will either; - Move from the reference cell to the test cell - E° is positive, A+ + e- A. Reduction in this cell; ½H2 H+ + e-. Oxidation in this cell - Move from the test cell to the reference cell - E° is negative, A- e- + A. Oxidation this cell; H+ + e- ->½H2. Reduction in this cell If E'° > 0 the electrons move to the test cell - The larger value the higher the driving force Hydrogen cell is just a reference - Any redox pair can be compared - The reduction potential for any redox reaction is given as: deltaE'° = E'° (electron acceptor) - E'° (electron donor) The actual E can differ a lot from the standard conditions - The E is calculated by the Nernst equation In the equation; - n is number of electrons transferred per molecule and F is the Faraday constant The free energy can be calculated as well: - deltaG= -nF deltaE or deltaG'° = -nF deltaE'°

Mutations in mitochondrial DNA

ROS is produced in mitochondria Mitochondrial DNA replication system is less efficient in reparing DNA damages Therefore defect mtDNA may accumulate over time - One ageing-theory says: accumulation of defects in mtDNA will cause the age symptoms Inheritance: - Animals inherit all their mitochondria from their mother - Only defects in mothers mtDNA will be passed to their offsprings - During the cell division the distribution of defective and non-defective mitochondria is uneven. - Heteroplasm

Free energy and equilibrium constants

Reactions goes towards equilibrium - Reactions are often reversible - Direction depends on concentrations - The equilibrium constant is the same regardless of the start conc. - At equilibrium the equilibrium constant of that reaction can be calculated - If the result of the division at start concentration is lower than the Keq of the reaction the reaction goes towards C and D - Is it higher at start concentrations it goes towards A and B - The further the reaction is from equilibrium, the higher the driving force; The magnitude of this driving force can be expressed as G

NAD-linked dehydrogenases - in oxidative phosphorylation

Reduced substrate + NAD^+ --> oxidized substrate + NADH + H^+ Remove two hydrogen atoms from their substrates - A hydride ion (:H-) to NAD+ - The other is released as H+ into medium NADH and NADPH are water-soluble electron cariers - Reversible associations to the dehydrogenases NADH cariers electrons from catabolic reactions to repsiratory chain NADPH supplies electrons to anabolic reactions Separate pools of NADH and NADPH - [Reduced form] / [oxidised form] high for NADPH and low for NADH

Reduction of the carbonyl group step 2 in fatty acid synthesis

Reduction of the carbonyl group at C3 position D-β-ketohydroxybutyryl-ACP is formed Catalyzed by the enzyme β-ketoacetyl-ACP reductase (KR) NADPH is the electron donor Note: during oxidation of fatty acids, L-β-hydroxy-acyl intermediates are formed

Mitochondrial complexes may associate in Respirasomes

Respirasomes - functional combinations of two or more electrons-transfer complexes Multienzyme Complex I and III can be isolated together if purification is done gently Multienzyme Complex III and IV can be observed in a complex by EM Kinetics support transfer of electrons through a tightly linked solid state The lipid Cardiolipin is especially abundant in inner mitochondrial memberane

Methyl group transfer

S-adenosyl methionine - Formed from ATP and methionine by release of Pi and PPi - Very reactive methyl group transfer - Is regenerated by methylation of homocysteine (coenzyme B12 or methyltetrahydrofolate)

ATP and (d)GTP, dTTP, UTP, (d)CTP

Several other (deoxy)nucleoside triphosphates exist - Have roughly the same amount of free energy as ATP - Known in DNA structure ((d)GTP, UTP, (d)CTP, dTTP) Only ATP is synthesized during catabolism - Transfer Pi to NDP to give NTP - Transfer PPi to NMP to give NTP - Costs no energy - Catalyzed by nucleoside diphosphate kinase

Generation of reactive oxygen species (ROS)

Several steps in the multienzyme complex pathway have potential to produce highly reactive free radicals (ROS) The radical •Q- are formed as an intermediate in the - Passage of electrons from QH2 to multienzyme Complex III - Passage of electrons from multienzyme Complex I to QH2 •^Q- may pass an electron to O2 to form •O2- (superoxide radical) This in term produces the even more reactive hydroxyl radical •OH The hydroxyl radical can then attack and damage anything it touches: proteins, lipids and DNA It is estimated the between 0.1- 4% of O2 used in respiration forms •^O2- .

Eicosanoids

Short ranging signaling molecules Release of ararchidonate Action of cyclooxygease (COX) / prostaglandin H2 synthase

Degradation of amino acids to acetyl-CoA

Six amino acids are degraded to acetyl-CoA: - Tryptophan - Lysine - Phenylalanine - Tyrosine - Leucine - Isoleucine

Degradation of amino acids to pyruvate

Six amino acids are degraded to pyruvate - Tryptophan - Alanine - Threonine - Glycine - Serine - Cysteine Methylene group is added via the folate-reaction Amino-transferase reaction

Sources of fatty acids

Sources 1 Fats consumed in the diet 2 Fats stored in the cells as lipid droplets - In vertebrates: adipose tissue/ adipocytes 3 Fats synthesized in one organ for export to another organ Nutritional guidelines - < 30% of the daily caloric intake from fat - In industrialized countries: ~40% of the daily energy from fat

Molecular mechanisms; specificity, amplification, desensitization/adaptation and integration.

Specificity; Signal molecule fits binding site on its complementary receptor; other signals do not fit. Amplification; When enzymes activate enzymes, the number of affected molecules increases geometrically in an enzyme cascade. Desensitization/adaption; Receptor activation triggers a feedback circuit that shuts off the receptor or removes it from the cell surface. Integration; When two signals have opposite effects on a metabolic characteristic such as the concentration of a second messenger X, or the membrane potential Vm, the regulatory outcome results from the integrated input from both receptors.

Oxidation of propionyl-CoA

Step 1 Propionyl-CoA is carboxylated - Formation of the D-stereoisomer of methyl-malonyl-CoA - Propionyl-CoA carboxylase - Biotin cofactor - CO2 is activated by attachment to biotin and then CO2 is transferred to the substrate - Formation of carboxy-biotin required for hydrolysis of ATP Step 2 Epimerization of D-methyl-malonyl-CoA - Formation of the L-stereoisomer of methyl-malonyl-CoA - Methylmalonyl-CoA epimerase Intramolecular rearrangement - Formation of Succinyl-CoA - Can enter the citric acid cycle - Methylmalonyl-CoA mutase - Coenzyme B12 (deoxyadenosyl-cobalamin) take part of the reaction - Derived from vitamin B12

Steps of fatty acid synthesis

Step 1 Transfer of acetyl-CoA to FAS: The acetyl group of acetyl-CoA is transferred to the Cys-SH group of β-ketoacyl-ACP synthase (KS) - Catalyzed by Malonyl/ Acetyl-CoA-ACP transacetylase (MAT). Step 2 Attachment of malonyl group: Transfer of malonyl group from malonyl-CoA to the -SH group of ACP - Catalyzed by malonyl/acetyl-CoA-ACP transferase (MAT)

Gluconeogenesis bypassing step 1 of glycolysis

Step 1 of the glycolysis Facilitated by glucose 6-phosphatase and Mg2+ Again, inorganic phosphate is released in an irreversible reaction

Steroid receptor

Steroid binding to a nuclear receptor protein allows the receptor to regulate the expression of specific genes

Fates of pyruvate

Still much chemical energy to harvest from pyruvate Three pathways for further metabolism - Decarboxylation to acetyl-CoA - Reduction to lactate - Decarboxylation and reduction to ethanol

The Citric Acid Cycle Step 6

Succinate is converted to fumarate - Reversible reaction - Production of FADH2 -As for NADH, electrons from FADH2 can also be transferred to O2 in the respiratory chain: Yields 1.5 ATP pr FADH2 (versus 2.5 from each NADH)

The Citric Acid Cycle Step 5

Succinyl-CoA is converted to Succinate - Facilitated by Succinyl-CoA synthetase -Synthetase: Joins two large molecules -Reverse reaction; joins succinate and CoA - CoA-SH is released - GTP is produced (from the energy of the thioester bond) - Reversible reaction The Reaction: - Succinyl-CoA synthetase binds to succinyl-CoA and replaces the CoA with inorganic phosphate - Upon release of succinate, the phosphoryl group in transferred to the enzyme - The phosphorylated enzyme transfers the phosphoryl to GDP, forming GTP (or ATP)

Biosynthesis of sphingolipids step 1 and 2

Synthesis of Sphinganine: 1 Combination of - Palmityl CoA - Serine by serine palmitoyltransferase 2 Reduction by Ketosphinganine reductase

ω-oxidation of fatty acids

Take place in endoplasmic reticulum (ER) Primary in liver and kidney fatty acids of 10 to 12 carbons Is relevant when β-oxidation is reduced Step 1: Introduction of a hydroxyl group at ω-carbon - Oxygen from O2 - Catalyzed by cytochrome P-450 enzymes (mixed function oxidases) - NADPH as an electron donor Oxidation: Alcohol dehydrogenase Step 2: 2nd oxidation: aldehyde dehydrogenase - NAD+ as an electron acceptor Attached by CoA - Performing β-oxidation - Forming succinate and adipic acid

Glycolysis step 4: Fructose 1,6-bisphosphate -> dihydroxyacetoneP and Glyceraldehyde (and the steps)

The 6-carbon ring is split into two 3-carbon chains (triose phosphates) by aldolase Reaction is reversible - because of low cellular concentration of products the deltaG is very low The conversion has several intermediate steps

Recap of regulation of the citric acid cycle and the glyoxylate cycle

The CAC is regulated at the entry point (PDH complex) and at step 1, 3 and 4 - In general low energy signals as ADP and AMP (and Ca2+) stimulates the activity while ATP inhibits it Citrate also regulates the committing step of the glycolysis, ensuring synchronization between the two pathways Vertebrates cannot direct fatty acids into the gluconeogenesis - Plants and some microorganisms can, as they have a glyoxysome, where fatty acids are converted into succinate -Succinate can feed the CAC and thus intermediates from here can be directed to the gluconeogenesis without depleting the CAC

Oxidation-Reduction Reactions in oxidative phosphorylation

The NADH dehydrogenase complex of the mitochondrial respiratory chain promotes the following series of oxidation-reduction reactions, in which Fe3+ and Fe2+ represent the iron in iron-sulfur centers, Q is ubiquinone, QH2 is ubiquinol, and E is the enzyme: (1) NADH + H+ + E-FMN → NAD+ + E-FMNH2 (2) E-FMNH2 + 2Fe3+ → E-FMN + 2Fe2+ + 2H+ (3) 2Fe2+ + 2H+ + Q → 2Fe3+ + QH2 Sum: NADH + H+ + Q → NAD+ + QH2 For each of the three reactions catalyzed by the NADH dehydroge-nase complex, identify (a) the electron donor, (b) the electron acceptor, (c) the conjugate redox-pair, (d) the reducing agent, and (e) the oxidizing agent.

Formation of carbamoyl phosphate

The NH4+ generated in the liver is used immediately An ATP-dependent reaction catalyzed by carbamoyl phosphate synthase I (in mitochondria). - Bicarbonate - 2 ATP Carbamoyl phosphate synthase II is another enzyme in the cytosol, taking part of the pyrimidine biosynthesis

Pyridoxal phosphate in aminotransferase

The aminotransferases have pyridoxal phosphate (PLP) as a prosthetic group Is formed from pyridoxine or vitamin B6 Is also found as coenzyme in the glycogen phosphorylase reaction Acts as an intermediate amino acid carrier at the active site of the amino transferases Pyridoxal phosphate under-goes reversible transforma-tions between - pyridoxal phosphate (aldehyde form) - pyridoxamine phosphate (aminated form) Pyridoxal phosphate is covalently bound to the active site of the enzyme through an aldimine linkage (Schiff-base)

Urea cycle in detail

The ammonia deposited in mitochondria of hepatocytes is converted to urea 1. Formation of citrulline - From ornithine and carbamoyl phosphate ornithine transcarbamoylase - Resembling the role of oxaloacetate in citric acid - Formed inside the mitochondria and citrulline is exported to the cytosol 2. The second amino group is introduced from aspartate - Aspartate is formed in mitochondria and transported to cytosol (2b) arginosuccinate synthetase - Require ATP by forming a citrullyl-AMP intermediate (2a) 3. The arginosuccinate is cleaved into fumarate and arginine arginosuccinate lyase - fumarate join the pool of citric acid intermediates 4.Arginine is cleaved to urea and ornithine. Arginase - Ornithine formed cytosolic is transported in to the mitochondria Will now enter a new round of urea cycle

Apoptosis and membrane

The asymmetric lipid distribution change then cells undergoes programmed cell death

Translocation of the butyryl group step 5 in fatty acid synthesis

The butyryl group is trans-located from the phospho-pantetheine-SH group on ACP to the Cys-SH group of β-ketoacyl-ACP synthase (KS) This was the position of the initial acetyl group

Catabolic pathway for asparagine and aspartate

The carbon skeletons of asparagine and aspartate enter the citric acid cycle as oxaloacetate The conversion of asparagine to aspartate is catalyzed by asparaginase Aspartate undergoes trans-amination with alpha-ketoglutarate to yield glutamate and oxaloacetate

Five amino acids are converted to alpha-ketoglutarate

The carbon skeletons of five amino acids enter citric acid cycle as alpha-ketoglutarate - Proline - Arginine - Glutamate - Glutamine - Histidine

Four amino acids are converted into succinyl-CoA

The carbon skeletons of four amino acids are degrade by pathways yielding succinyl-CoA - Methionine - Isoleucine - Threonine - Valine

The Citric Acid Cycle Step 1

The carbonyl of oxaloacetate acts as an electrophilic center - Attacked by the methyl center of acetyl-CoA - Claisen condensation - CoA-SH released (reused in another Pyruvate -> Acetyl-CoA) Facilitated by citrate synthase - Highly exergonic (irreversible) reaction (hydrolysis of thioester) Citrate synthase is a homodimer - Two identical subunits - Performs two reaction at the time - Undergo large conformational change upon binding

Formation of Malonyl-CoA

The carboxyl group is first transferred to the biotin group - ATP-depended reaction The biotinyl transfer the CO2 to acetyl-CoA in the second step

Tetrahydrofolate

The cofactor is synthesized in bacteria Folate is a vitamin for mammals The one-carbon group undergoing transfer, in any three oxidation states bonded to N-5 or N-10 or both

Glycogen debranching enzyme

The debranching enzymes transfers three of the four residues to the adjoining string It also removes the last debranching residue - Breaks the alpha1→6 link by hydrolysis - Non-phosphorylyzed glucose is released Glycogen phosphorylase continues on the new unbranched chain

Actual free energy

The deltaG'^o is for comparison and simplicity - Describes the reaction at a set of specific conditions (table value) - In the cells, the reactions occur at conditions far from standard - The ACTUAL deltaG must be calculated for each specific condition and changes as the reaction proceeds (dependent on concentrations) The reaction is spontaneous only if deltaG is negative, regardless of deltaG'^o - deltaG'ô can easily be negative, while deltaG is positive e.g. if reactant concentrations are very low; [A]*[B] ≈ 0, -> ln(Q) is high and positive -> RT ln(Q) > deltaG'^o

Reduction of the double bond step 4 in fatty acid synthesis

The double bond of trans-Δ2-butenoyl-ACP is reduced Butyryl-ACP is formed The reaction is catalyzed by enoyl-ACP reductase (ER) NADPH is the electron donor

Addition of two carbons to the growing fatty acyl chain

The fatty acid carbon chain is formed by a repeating four step sequence The produced saturated acyl group is substrate for an additional round of condensation

Oxidation sted of carbon

The fewer electrons 'owned' by C the higher the oxidation step

Recap of Regulation of Metabolic pathways

The flow of metabolites are controlled by the activity of enzymes Enzyme activity can be regulated in two main ways (10 subgroups) - Quantitatively - in/decrease of the number of enzymes - Qualitatively - in/decrease in the specific enzymatic activity - Regulation often occurs in several of the 10 groups at a time Often regulation takes place at the first step of a metabolic pathway - Regulatory steps have high negative ∆G AMP level is an important signal for low ATP levels - Activates AMPK, leading to energy saving signals and ATP synthesis

Sequence of electron carriers

The general flow of electrons is: 1. (NADH or succinate) to Flavoproteins (i.e. proteins containing FMN/FAD) 2. Flavoproteins to Ubiquinone 3. Ubiquinone to iron-sulfur proteins 4. Iron-sulfur proteins to cytochromes 5. Cytochrome to O2

Regulation of glycolysis/gluconeogenesis Liver

The glycolysis is also tightly regulated at the final step - First step of the gluconeogenesis - PEP ↔ Pyruvate Molecules indicating high energy inhibits the pyruvate kinase - ATP, acetyl-CoA, long-chain fatty acids (and alanine product of pyruvate) Fructose 1,6-Bisphosphate stimulate pyruvate production The liver isoform of Pyruvate kinase is also regulated by phosphorylation (inhibition, by glucagon) - Prevents liver from using glucose at low blood sugar

Insulin and glucagon in Regulation of glycolysis/gluconeogenesis

The hormones insulin and glucagon regulates the glycolysis and gluconeogenesis - Insulin activates PFK-2 and inactivates FBPase-2 - Glucagon has the opposite effect

Regulation of carbamoyl phosphate synthase I

The level of NH4+ may vary Animals on protein-free diet has lower levels of the urea cycle enzymes Allosteric regulation of carbamoyl phosphate synthase I - Activation by N-acetylglutamate - Formed from acetyl-CoA and glutamate - Regulated by arginine - In mammals, N-acetylglutamate is only formed in liver

Main entry of NH4+

The main entry into biomolecules is Glutamate + NH4+ ATP --> Glutamine + ADP + Pi + H+ - Catalyzed by Glutamine Synthetase In bacteria and plants, Glutamate is syntesized from α-ketoglutarate by glutamate synthase: α-ketoglutarate + glutamine + NADPH + H+ --> 2 L-glutamate + NADP+ THE NET REACTION - SUM of glutamate synthesis is shown in the picture. In animals, L-glutamate is obtained through trans-amination

Muscle versus liver responses in metabolic regulation

The metabolic regulation of carbohydrates differ in three main ways in the myocytes compared to the hepatocyte Myocytes uses its glycogen only for own needs - No glucose is released to the blood - They have no glucagon receptors The pyruvate kinase isoform in myocytes is stimulated by increased cAMP levels - Hepatocytes are inhibited by cAMP to prevent them from using glucose during low energy levels (saved for the brain/muscle) Myocytes do not have the enzymes for glyconeogenesis

Homeostasis

The metabolism is a complex web of intertwined pathways - 30,000 different proteins in a cell - Thousands of different reactions are catalyzed - Hundreds of different metabolites are involved Flow direction, flow rate and concentrations of metabolites constantly needs to be controlled At physiological changes (due to exercise, intake of food etc.) the reactions need to be regulated It is crucial that some key molecules are kept at a constant concentration - Blood sugar (brain), ATP, H+ (pH) - The maintenance of this dynamic steady state is called homeostasis Discriminate between: - Metabolic regulation: Maintain homeostasis - Metabolic control: Change of pathways in response to external conditions

Dephosphorylation - enzyme regulation

The most common regulatory mechnism. Occur within seconds or minutes of the signal - Signal is usually extracellular

Regulation of glycolysis/gluconeogenesis in different tissues

The need for glucose differs among tissues - Different regulations of the glycolysis/gluconeogenesis Four different isozymes of hexokinase exists - Same substrate and product - Different catalytic activities - Especially type IV (liver) differs from I-III (muscle) Type I-III: - very low Km - Fully saturated almost always - Inhibited by glucose 6-P (product) Type IV: - High Km - Very dependent on glucose level -High after a meal (high blood sugar) -Low activity at fasts (low blood sugar) Gluconeogenesis takes over - ↑blood sugar - Not inhibited by glucose 6-P

deltaG

The numerical value of G is dependent on: Temperature, Pressure, Starting concentrations The term deltaG^o (standard) is introduced in the book(table values) are always at 25,C1 ATM, 1 M of each reactant and product. Biochemist have a further standardization deltaG'^o (transformed) - pH 7 (H+ is 10-7 M and not 1 M, pH 0) - Water is 55.5 M not 1 M - Mg2+ is often set at 1 mM - When H2O, Mg2+ or H+ are reactants or products, they are not included in the calculations but are incorporated in the values of K'eq and deltaG'^o A deltaG'^o value can be obtained for each reaction under standard conditions deltaG'^o = -RT * lnK'eq

Pyruvate dehydrogenase complex

The overall reaction - Three different types of subunits (enzyme complex) - Five cofactors - Irreversible

Glycolysis vs Gluconeogenesis

The overall reaction of the glycolysis: Glucose + 2 ADP + 2 Pi + NAD+ → 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ 2 H2O - Produces 2 high energy phosphate groups and 2 NADH The overall reaction of the Gluconeogenesis: 2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 4 H2O → Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ - Costs 6 high energy phosphate groups and 2 NADH Necessary to ensure irreversibility; unidirectional flow; If glycolysis and gluconeogenesis were running at the same time, it would be wasting energy

Action of plant desaturase in fatty acid biosynthesis

The plant desaturase is located in ER and chloroplast Act on phospholipids not the free fatty acids Essential fatty acids in mammals - Linolate (18:2(Δ9,12)) - α-linolenate (18:3(Δ9,12,15)) Converted to - γ-linolenate (18:3(Δ6,9,12)) - Eicosatrienoate (20:3(Δ8,11,14)) - Arachidonate (20:4(Δ5,8,11,14)) Fatty acid elongation system

Transport of lipids - chylomicron

The protein moieties acts as signals in the uptake and metabolism of chylomicron contents Triacylglycerols make up more than 80% of the mass The diameter ranges from 100 - 500 nm.

V-class proton pump mechanism

The proton pumping by the vesicular (V) -class can generate electric potential or H+ concentration gradients

Glycolysis step 10: PEP → Pyruvate

The pyruvate kinase facilitates the final step of the glycolysis Transfers a phosphoryl group to ADP in the presence of Mg2+ and K+ Energy release is conserved in ATP - Still 2 molecules from the starting glucose Irreversible Concentrations of pyruvate of the enol form are kept low Driving force is high - more energy is released Ensures the high negative deltaG, making the reaction irreversible

Recap of Metabolic control and Coordinated regulation of Glycolysis and Gluconeogenesis

The response coefficient (R) is the product of its elasticity (ε) and flux coefficient (C) As the glucose needs differs in cell types, they have different isoforms of some of the metabolic enzymes Pathways can usually go in both directions - Never at the same time - ensured by reciprocal regulation The committing step of the glycolysis (F6-P ↔ F1,6-BP) is tightly regulated - the key regulator is F2,6-BP - So is the 10th step (first step of gluconeogenesis) Enzyme number is regulated by hormones via stimulation/inhibition of transcription factors

Gluconeogenesis bypassing irrversible steps

The three irreversible steps from glycolysis to be bypassed is 1, 3, and 10.

The nitrogen cycle

The total amount of nitrogen fixed annually in the biosphere exceeds 10^11 kg. Reactions with red arrows occur largely or entirely in anaerobic environments. The redox states of the various nitrogen species are depicted at the bottom of the figure.

Glycolysis step 5: DihydroxyacetoneP -> Glyceraldehyde 3-P

The two triose phosphates are made a like by triose phosphate isomerase in a reversible step Only glyceraldehyde 3-P is used further in the glycolysis pathway Reversible

Metabolic pathways

There are several 'steps' or conversions - A change of the metabolite, is often assisted by an enzyme Transformation from start product to end product Different start products can have the same end product - Convergent - usually catabolic pathway One start product can have several end products (different pathways) - Divergent - usually anabolic pathway Cyclic pathways also exists

Regulatory responses carbohydrate metabolism

There are three main regulatory responses in regards of carbohydrate metabolism: - After a meal: Blood sugar rises. Insulin is released. Stimulates cells to take up glucose and store it as glycogen - During fasts: Low blood sugar. Glucagon is released. Stimulates liver cells to break down glycogen to glucose and release it to the blood. Liver pyruvate kinase in inactivated, preventing glucose uptake - Fight or flight response: Sudden strenuous exercise. Epinephrine is released. Muscle cells converts glycogen to glucose for ATP. Picture is the insulin/glucagon interplay in a more detailed view - Main initiator is the blood sugar - Note the reciprocal regulation

Thioesters and oxygen esters (in changing the deltaG)

Thioesters are compounds with high deltaG when hydrolyzed. - Important in metabolism - Most common is Acetyl-CoA Oxygen esters resonate to a compound not subjectable to hydrolysis. - Leads to a decrease in reactants Raises the deltaG (closer to zero)

Cellular respiration

Three main stages: - Production of acetyl-CoA; Not only glucose: - Amino acids - Fatty acids Oxidation of acetyl-CoA - CO2 - NADH/FADH2 Respiratory chain e- is used to convert H+ and O2 to H2O while ADP + Pi is converted to ATP: 1 H- from NADH = 2.5 ATP

Oxidation of Fatty Acids

Three stages Stage 1: b-oxidation of fatty acids: Successive removal of two-carbon units as acetyl-CoA Removal of four hydrogen atoms Stage 2: Citric acid cycle Oxidation of acetyl-CoA to CO2 Stage 3: Respiratory chain: Oxidation of NADH and FADH2 in the respiratory chain

Electron transfers - in oxidative phosphorylation

Three types of electron transfers 1. Direct transfer (Fe3+ + e- → Fe2+) 2. Transfer as a hydrogen atom (or H+ and e-) 3. Transfer as a hydride (:H-) Reducing equivalent = transfer of a single electron Three electron carriers in respiratory chain other than FAD and NAD+: - Ubiquinone / coenzyme Q (a hydrophobic quinone) - Heme-type iron containing proteins - Fe-S type iron containing proteins

Phosphoglucomutase

To enter the glycolysis (or gluconeogenesis), glucose 1-P must be converted to glucose 6-P Two step transition - Phosphate group is transferred to C-6 - Reacts with C-1 phosphate before release

Role of Pyridoxal phosphate (PLP)

Transamination - Amino acids - Aminosugars (Ex. perosamine) Racemization Decarboxylation - Amino acids β-elimination - Serine dehydratase

Group transfers between two nucleophiles (metabolism)

Transfer between two nucleophiles

Group transfers phosphoryl group (metabolism)

Transfer of a phosphoryl group (e.g. from ATP), which leaves easily to an OH group. - The receiver is 'activated', making reactions with other molecules easier.

The Citric Acid Cycle Step 3

Transformation of isocitrate to α-ketoglutarate can be divided into three steps - Only step one is enzyme facilitated: Isocitrate dehydrogenase transfers hydride to either NAD or NADP: Oxalosuccinate - The electron withdrawal leads to decarboxylation (Mn2+ assisted) - Rearrangement and addition of a proton leads to α-ketoglutarate - ∆G'° = -20.9 kJ/mol

Carbon cycle of Citric Acid Cycle

Two carbons enter the CAC as acetate In two steps of the CAC CO2 is released - However, following the carbons in the cycle reveals that it is not the same carbons entering the CAC that leaves during the cycle

Biosynthesis of membrane lipids

Two classes of membrane phospholipids - Glycerophospholipids - Sphingolipids Attaching phospholipid head groups - Phosphorylation of phosphatidic acid

The Citric Acid Cycle Step 2

Two-step reaction by aconitase - Dehydration of citrate to cis-aconitate - Rehydrated to isocitrate - Isomerization - aconitase is an isomerase Reversible reaction - Rapid consumption of isocitrate in the next step

The allosteric effector effect - enzyme regulation

Typically converts hyperbolic kinetics to sigmoid curves At steep points, small [S] changes can mean large changes in activity The allosteric effect can be expressed by the hill coefficients, nH (chapter 5) nH < 1 = Negative cooperativity nH > 1 = positive cooperativity

Citric Acid Cycle regulation

Under regulation at several steps - Especially the flow into the cycle is tightly regulated by several sterical modulators - Stimulating factors: -Molecules indicating low energy levels -Ca2+ released in muscles upon contraction also stimulates the process as it is a sign of ATP consumption Inhibitory factors: - Complementary signals to those above - high energy - Fatty acids is converted to Acetyl-CoA as well through another pathway - preferred energy source Also controlled at several irreversible steps within the cycle - Step 1,3 and 4 - Can become rate-limiting steps - They are mainly regulated by products and -intermediates - High NADH/NAD+ ratio inhibits rate of reactions producing NADH The CAC-flow match the flow of glycolysis - Only the amount of pyruvate needed in CAC is formed in the glycolysis - Citrate inhibits PFK-1: Glycolysis ↓ The regulated intermediates is also branching points for the precursors for many biosynthesis pathways - Enables regulation of their pathways Reactions often takes place in metabolons - Multi-enzyme complexes -Efficient passage - higher reaction rate -Substrate channeling

Glycerol 3-phospahte shuttle

Using cytosolic glycerol 3-phosphate dehydrogenase Forms Glycerol 3-Phosphate Mitochondrial glycerol 3-phosphate dehydrogenase transfer electrons to FAD and further to ubiquinone Only get 1.5 ATP

Definition of metabolism

Utilization of food (energy rich nutrients) (autotrophs get their carbon from the air) - Starch/glycogen - Fat - Protein Breakdown to monomers - 'building blocks'/precursors (obatin energy) - Monosaccharides - Acetyl Coenzyme A - Amino acids Synthesis of cell specific molecules (used for cellular growth and maintenance, uses energy) - Glycogen - Lipids - Proteins - DNA/RNA - Etc

Acyl-CoA synthases

Various isoenzymes: - short, intermediate or long carbon chains Catalyze formation of the thioester linkage Coupled to the cleavage of ATP to AMP and PPi The reaction occur in two steps deltaG'0 for the hydrolysis of Acyl-CoA is ~ -31 kJ/mol

The glyoxylate cycle

Vertebrates cannot convert fatty acids into carbohydrates PEP carboxykinase can convert oxaloacetate into PEP for gluconeogenesis - Will deplete the oxaloacetate - No net production - CAC will come to a halt When fatty acids enter the cycle, they can only be used for precursors or energy - cannot go into the glycolysis Plants and some microorganisms can convert acetate into PEP (which enters gluconeogenesis) - They have the glyoxylate cycle - Acetyl is converted into succinate -Feed the CAC with succinate -Converted to oxaloacetate (->PEP) 2Acetyl-CoA + NAD+ + 2H2O --> Succinate + 2CoA + NADH + H+ Vertebrates lack isocitrate lysate and malate synthase Occur in glyoxomes - Common enzymes for CAC and Glyoxylate cycle are different isoforms Fatty acids are converted to succinate (Glyoxylate cycle) Succinate enters the CAC Exits mitochondria as malate Converted to hexose As the CAC is fed with intermediates, they can be converted to sugars without exhausting the CAC

Fructose 2,6-Bisphosphate

Very potent allosteric regulator of both PFK-1 and FBPase-1 The F26BP level is regulated by PFK-2 and FBPase-2

Stoichiometry of ATP Production: Effect of c Ring Size (Q)

a) Bovine mitochondria - If the c ring has 8 c subunits, then one full rotation will transfer 8 protons to the matrix and produce 3 ATP molecules. - But this synthesis also requires the transport of 3 Pi into the matrix, at a cost of 1 proton each, adding 3 more protons to the total number required. - This brings the total cost to (11 protons)/(3 ATP) = 3.7 protons/ ATP. - The consensus value for the number of protons pumped out per pair of electrons transferred from NADH is 10 - So, oxidizing 1 NADH produces (10 protons)/(3.7 protons/ATP) = 2.7 ATP. b) Yeast mitochondria - If the c ring has 10 c subunits, then one full rotation will transfer 10 protons to the matrix and produce 3ATP molecules. - 3 protons to transport the 3 Pi into the matrix - The total cost is 13 protons/ 3 ATP = 4.3 protons/ATP. - Oxidizing 1 NADH produces 10 protons giving 10 protons/ 4.3 protons/ATP = 2.3 ATP. What are the comparable values for electrons entering the respiratory chain from FADH2? - Electrons from FADH2 only 6 protons that are available to drive ATP synthesis. - This changes the calculation for bovine mitochondria to a) (6 protons)/ (3.7 protons/ATP) = 1.6 ATP per pair of electrons from F ADH2. - For yeast mitochondria, the calculation is b) (6 protons)/(4.3 protons/ATP) = 1.4 ATP per pair of electrons from FADH2. - These calculated values of x or the P/O ratio define a range that includes the experimental values of 2.5 ATP/NADH and 1.5 ATP/FADH2.

Second level regulation of glutamine synthetase

a) an adenylated Tyr residue. b) Cascade leading to adenylation (inactivation) of glutamine synthetase. AT represents adenylyltransferase; UT uridylyltransferase. PII is a regulatory protein, itself regulated by uridylylation. (detaljer side 890)

Mechanisms of lipid transport

a; lateral movement occurs between the two leaflets of each organelle membrane. b; transbilayer movement or flip-flop - as lipids form the backbone of a membrane, they are an integral part of vesicular carriers (c; vesicular transport) that connect, for example, the endoplasmic reticulum (ER) and the plasma membrane indirectly through the Golgi (not shown). At the same time, lipids can be exchanged as monomers between the cytosolic surfaces of organelle membranes. d; monomeric exchange - Monomeric exchange is especially relevant for organelles such as mitochondria and chloroplasts that are not connected to other organelles by vesicular pathways, and can be facilitated by lipid-transfer proteins. Maintaining the differences in lipid composition between organelles and between the two leaflets of individual organelle membranes requires selectivity and directionality in the various transport mechanisms

Bioenergetics

is the quantification of energy - Energy yield of nutrients - Energy spend on processes - Likelihood of processes to occur spontaneous;High energy state > Low energy state - Understanding the direction of the flow

The Citric Acid Cycle Step 4

α-ketoglutarate is converted into succinyl-CoA - α-ketoglutarate dehydrogenase complex - Irreversible - Conversion of NAD+ to NADH, release of CO2 - Virtually the same reaction as the PDH (Pyruvate -> Acetyl-CoA); Same cofactors

Mitochondrial defense of ROS formation

•^O2^- is removed by superoxide dismutase, glutathione peroxidase, and glutathione reductase Superoxide dismutase : 2•^O2^- + 2H+ → H2O2 + O2 Glutathione peroxidase: H2O2 + Glutathione (red) → H2O + Glutathione (oxid) Glutathione reductase: Glutathione (oxid) + NADPH → Glutathione (red) + NADP+ Nicotinamide nucleotide transhydrogenase: NADP+ + 2H → NADPH + H+