Biochem Final

Chapter 13 Principle 1

The chemical changes and energy transductions in living organisms follow the laws of thermodynamics.

Theoretical vs Actual Free Energy Changes

Theoretical: -Free Energy change under standard condition is -30.5 kJ/mol -But this is for ATP → ADP + Pi Actual (varies from cell to cell): -Free Energy change for MgATP2- hydrolysis is -51.5 kJ/mol -Much more exergonic than the theoretical value! Defined as the phosphorylation Potential (∆GP ) = actual energy available for biochemical reactions For simplicity's sake we will usually use the theoretical value (-30.5 kJ/mol)

Sequential Reactions in Gluconeogenesis

*see chp 14 slide 101*

Regulation of Glycolysis and Gluconeogenesis by Fructose 2,6-Bisphosphate

*see chp 14 slide 118 for graphs and equation*

Entry of Dietary Glycogen, Starch, Disaccharides, and Hexoses into the Preparatory Stage of Glycolysis

*see chp 14 slide 54*

Stoichiometry of Coenzyme Reduction and ATP Formation in Aerobic Oxidation of Glucose

*see chp 16 slide 69*

Relationship Between Catabolism and Anabolism

*see image*

In Eukaryotes, the Mitochondrion Is the Site of Energy-Yielding Oxidative Reactions and ATP Synthesis

---isolated mitochondria contain all enzyme, coenzymes, and proteins needed for: - the citric acid cycle - electron transfer and ATP synthesis by oxidative phosphorylation - oxidation of fatty acids and amino acids to acetyl-CoA - oxidative degradation of amino acids to citric acid cycle intermediates

Hydrolysis of Disaccharides

--membrane-bound hydrolases in the intestinal brush border hydrolyze disaccharides: -Dextrin + nH20 --dextrinase--> nGlucose -Maltose + H20 --Maltase--> 2 Glucose -Lactose + H20 --Lactase--> Galactose + Glucose -Sucrose + H20 --Sucrase--> Fructose + Glucose -Trehalose + H20 --Trehalase--> 2 Glucose -monosaccharides pass through intestinal cells to the bloodstream, which transports them to the liver or other tissues

Phosphofructokinase-1 (PFK-1) and Fructose 1,6- Bisphosphatase Are Reciprocally Regulated

-ATP inhibits PFK-1 by binding to an allosteric site -ADP and AMP allosterically relieve this inhibition by ATP

The Special Role of ATP

-ATP is the chemical link between catabolism and anabolism -energy obtained from catabolism of nutrient molecules is used to make ATP from ADP and Pi -the exergonic conversion of ATP to ADP and Pi (or to AMP and PPi ) is coupled to many endergonic reactions and processes

Chapter 13 Principle 4

-ATP is the universal energy currency in living organisms. --Transfer of its phosphoryl group to a water molecule or metabolic intermediates provides the energetic push for muscle contraction, the pumping of solutes against concentration gradients, and the synthesis of complex molecules.

Actual Free-Energy Change

-Depend on Reactant and Product Concentrations for any reaction aA + bB ⇄ cC + dD, ∆G and ∆G′° are related by the equation: *see image* -rightmost part is the mass-action ratio (Q) -When ∆G = 0, the system is at equilibrium

Chapter 16 Principle 5

-Enzymes have evolved to form complexes to efficiently achieve a series of chemical transformations without releasing the intermediates into the bulk solvent. -This strategy, seen in the pyruvate dehydrogenase complex and the metabolons of the citric acid cycle, is ubiquitous in other pathways of metabolism, in respiration, and in the many " — somes" that assemble and disassemble informational macromolecules.

Chapter 14 Principle 4

-Gluconeogenesis is the synthesis of glucose from simpler precursors like pyruvate and lactate. -Although it uses seven of the ten enzymes that also act in glycolysis, gluconeogenesis must bypass three of the most exergonic steps in glycolysis with energetically favorable reactions unique to gluconeogenesis

Chapter 14 Principle 2

-Glucose and other hexoses and hexose phosphates obtained from stored polysaccharides or dietary carbohydrates feed into the glycolytic pathway. -By using a common pathway for a number of starting materials, the cell economizes on the number of enzymes that must be synthesized and simplifies the regulation of the common pathway.

Why glucose stays inside the cell against a concentration gradient...

-Glucose is kept inside the cell by conversion to glucose 6-phosphate. -Phosphorylation traps glucose inside the cell because the phosphate group has a negative charge at physiological pH.

Chapter 14 Principle 5

-Glycolysis and gluconeogenesis are reciprocally regulated so that both processes don't occur simultaneously in a futile cycle. -Most regulatory mechanisms act on reactions that are unique to each pathway.

Glycolysis

-Glycolysis is an Almost Universal Central Pathway of Glucose Catabolism -glycolysis = process by which a molecule of glucose is degraded in a series of enzyme-catalyzed reactions to yield two molecules of the three-carbon compound pyruvate; some free energy is conserved as ATP and NADH An Overview of Glycolysis: *chp 14 slide 14* An Overview: Glycolysis Has Two Phases: --preparatory phase: - ATP is consumed - hexose carbon chains are converted to glyceraldehyde 3-phosphate --payoff phase: -yields energy conserved as 2 ATP and 2 NADH and 2 pyruvate -each of the two molecules of glyceraldehyde 3- phosphate undergoes oxidation at C-1 -some energy from the oxidation reaction is conserved in the form of one NADH and two ATP per triose phosphate oxidized ATP and NADH Formation are Coupled to Glycolysis: --the overall equation for glycolysis is: - glucose + 2NAD+ + 2ADP + 2Pi ⟶ 2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O Resolving the Equation of Glycolysis into Two Processes: --the conversion of glucose to pyruvate is exergonic: -glucose + 2NAD+ ⟶ 2 pyruvate + 2NADH + 2H+... ∆G′°1 = −146 kJ/mol --the formation of ATP from ADP and Pi is endergonic: -2ADP + 2Pi ⟶ 2ATP + 2H2O... ∆G′°2 = 2(30.5 kJ/mol) = 61.0 kJ/mol The Standard Free-Energy Change of Glycolysis (∆G′° Sum): --the sum of the two processes gives the overall standard free-energy change of glycolysis, ∆G′° Sum: - ∆G′° Sum = ∆G′° 1 + ∆G′° 2 = −146 kJ/mol + 61.0 kJ/mol. = −85 kJ/mol -under standard and cellular conditions, glycolysis is essentially irreversible The Preparatory Phase of Glycolysis Requires ATP: --in the preparatory phase of glycolysis: -two molecules of ATP are invested to activate glucose to fructose 1,6- bisphosphate - the bond between C-3 and C-4 of fructose 1,6-bisphosphate is then broken to yield two molecules of triose phosphate -*see chp 14 slide 22* 10 STEPS OF GLYCOLYSIS: Step 1. Phosphorylation of Glucose: --hexokinase activates glucose by phosphorylating at C-6 to yield glucose 6-phosphate -ATP serves as the phosphoryl donor -hexokinase requires Mg2+ for its activity -irreversible under intracellular conditions *see chp 14 slide 23* Step 2. Conversion of Glucose 6- Phosphate to Fructose 6-Phosphate: --phosphohexose isomerase (phosphoglucose isomerase) catalyzes the reversible isomerization of glucose 6-phosphate (an aldose) to fructose 6-phosphate (a ketose) -reaction readily proceeds in either direction *see chp 14 slide 27 and slide 28* Step 3. Phosphorylation of Fructose 6- Phosphate to Fructose 1,6-Bisphosphate: --phosphofructokinase-1 (PFK-1) catalyzes the transfer of a phosphoryl group from ATP to fructose 6-phosphate to yield fructose 1,6-bisphosphate -essentially irreversible under cellular conditions -the first "committed" step in the glycolytic pathway *see chp 14 slide 29* --Allosteric Regulation of Phosphofructokinase-1: -activity increases when: ATP supply is depleted and/orADP and AMP accumulate -fructose 2,6-bisphosphate is a potent allosteric activator -ribulose 5-phosphate indirectly activates Step 4. Cleavage of Fructose 1,6-Bisphosphate: --fructose 1,6-bisphosphate aldolase (aldolase) catalyzes a reverse aldol condensation and cleaves fructose 1,6-bisphosphate to yield glyceraldehyde 3- phosphate and dihydroxyacetone phosphate -reversible because reactant concentrations are low in the cell *see chp 14 slide 31* Step 5. Interconversion of the Triose Phosphates: --triose phosphate isomerase converts dihydroxyacetone phosphate to glyceraldehyde 3-phosphate -reversible -final step of the preparatory phase of glycolysis *see chp slide 32* -after step 5 of glycolysis, the carbon atoms derived from C-1, C-2, and C-3 of the starting glucose are chemically indistinguishable from C-6, C-5, and C-4, respectively (*see slide 33*) Step 6. Oxidation of Glyceraldehyde 3-Phosphate to 1,3-Bisphosphoglycerate: --glyceraldehyde 3- phosphate dehydrogenase catalyzes the oxidation glyceraldehyde 3- phosphate to 1,3- bisphosphoglycerate -energy-conserving reaction *see chp 14 slide 36* --The First Step of the Payoff Phase is an Energy-Conserving Reaction: -formation of the acyl phosphate group at C-1 of 1,3- bisphosphoglycerate conserves the free energy of oxidation -acyl phosphates have a very high standard free energy of hydrolysis (∆G′° = −49.3 kJ/mol) --The Glyceraldehyde 3-Phosphate Dehydrogenase Reaction: *see chp 14 slide 38* Step 7. Phosphoryl Transfer from 1,3-Bisphosphoglycerate to ADP: --phosphoglycerate kinase transfers the high-energy phosphoryl group from the carboxyl group of 1,3- bisphosphoglycerate to ADP, forming ATP and 3- phosphoglycerate -*see chp 14 slide 39* --Note that Steps 6 and 7 of Glycolysis Constitute an Energy-Coupling Process: -the sum of the two reactions is: glyceraldehyde 3-phosphate + ADP + Pi + NAD+ ⇄ phosphoglycerate + ATP + NADH + H+ ... ∆G′° = −12.2 kJ/mol -substrate-level phosphorylation = the formation of ATP by phosphoryl group transfer from a substrate (different from respiration-linked phosphorylation) Step 8. Conversion of 3- Phosphoglycerate to 2- Phosphoglycerate: --phosphoglycerate mutase catalyzes a reversible shift of the phosphoryl group between C-2 and C-3 of glycerate -requires Mg2+ *see chp 14 slide 41* --The Phosphoglycerate Mutase Reaction: *see chp 14 slide 42 and 43 Step 9: Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate: --enolase promotes reversible removal of a molecule of water from 2-phosphoglycerate to yield phosphoenolpyruvate (PEP) -energy-conserving reaction -mechanism involves a Mg2+-stabilized enolic intermediate *see chp 14 slide 46* Step 10: 2nd Production of ATP by Pyruvate Kinase: --pyruvate kinase catalyzes the transfer of the phosphoryl group from phosphoenolpyruvate to ADP, yielding pyruvate -requires K+ and either Mg2+ or Mn2+ *see chp 14 slide 47* --Pyruvate in its Enol Form Spontaneously Tautomerizes to its Keto Form: -pyruvate kinase catalyzes the transfer of the phosphoryl group from phosphoenolpyruvate to ADP, yielding pyruvate -requires K+ and either Mg2+ or Mn2+ *see chp 14 slide 48* Overall glycolysis Balance Shows a Net Gain of Two ATP and Two NADH Per Glucose: -subtracting the two ATP spent in the preparatory phase, the net equation for the overall process is: glucose + 2NAD+ + 2ADP + 2Pi ⟶ 2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O

Energetics of Some Chemical Reactions

-Hydrolysis reactions are generally strongly favorable or spontaneous (as opposed to condensation reactions) -Isomerization reactions have smaller free energy changes (∆G° = 0 or near 0) -Complete oxidation of reduced compounds is strongly favorable (But in biochemistry, these reactions are highly regulated)

2) Internal Rearrangements, Isomerizations, and Eliminations

-Internal rearrangements and isomerizations: redistribution of electrons results in alterations without changes in the overall oxidation state of the molecule -In biochem, elimination reactions usually involve the loss of a water group. reactions include: -intramolecular oxidation-reduction -change in cis-trans arrangement at a double bond -transposition of double bonds *in image. A = isomerization. Bottom = elimination

In cells, ATP and ADP form a complex with Mg2+

-Mg2+ in the cytosol binds to ATP and ADP -for most enzymatic reactions involving ATP, the true substrate is MgATP2 *see chp 13 slide 34 for image*

Chapter 13 Principle 5

-Oxidation-reduction reactions indirectly provide much of the energy needed to make ATP. --Reduced substrates such as glucose are oxidized in several steps, with the energy of oxidation steps conserved in the form of a reduced cofactor, NADH. --Energy stored in NADH is used to drive the synthesis of ATP.

Chapter 16 Principle 1

-Pyruvate is the metabolite that links two central catabolic pathways, glycolysis and the citric acid cycle. -It is therefore a logical point for regulation that determines the rate of catabolic activity and the partitioning of pyruvate among its possible uses

free energy change

-The balance between enthalpy, entropy and temperature for a process -If delta G is negative, the reaction is spontaneous; irreversible -If delta G is positive, the reaction requires initial energy input

Chapter 16 Principle 4

-The central role of the citric acid cycle in metabolism requires that it be regulated in coordination with many other pathways. Regulation occurs by both allosteric and covalent mechanisms that overlap and interact to achieve homeostasis. -Some mutations that affect the reactions of the citric acid cycle lead to tumor formation

Chapter 16 Principle 3

-The citric acid cycle is a hub of metabolism, with catabolic pathways leading in and anabolic pathways leading out. -Acetate groups (acetyl-CoA) from the catabolism of various fuels are used in the synthesis of such metabolites as amino acids, fatty acids, and sterols. -The breakdown products of many amino acids and nucleotides are intermediates of the cycle, and they can be fed in or siphoned off as needed by the cell.

Chapter 13 Principle 2

-The free-energy change is the maximum energy made available to do work when a chemical reaction occurs. --If two reactions can be combined to yield a third reaction, the overall free energy change is the sum of the two. --Cells accomplish energy-requiring chemical work by coupling an energy-releasing (exergonic) reaction, such as the cleavage of ATP, to an endergonic reaction (which requires energy input).

Chapter 14 Principle 6

-The pentose phosphate pathway is an alternative pathway for glucose oxidation. -It yields pentoses for nucleotide synthesis and reduced cofactors for biosynthesis of fatty acids, sterols, and many other compounds.

Chapter 16 Principle 2

-The reactions of the citric acid cycle follow a chemical logic. -In its catabolic role, the citric acid cycle oxidizes acetyl-CoA to CO2 and H2O. -Energy from the oxidations in the cycle drives the synthesis of ATP. -The chemical strategies for activating groups for oxidation and for conserving energy in the form of reducing power and high-energy compounds are used in many other biochemical pathways

In brewing beer, why is it necessary to use an enclosed container so O2 can be excluded?

-Under aerobic conditions, yeast oxidizes the product of glycolysis to CO2 and H2O. When supplied with oxygen, yeast oxidizes the pyruvate formed by glycolysis to CO2 and H2O via the citric acid cycle. -When all the dissolved oxygen in the vat has been consumed, the yeast cells switch to anaerobic metabolism and ferment the sugars into ethanol and CO2

Thermodynamically Unfavorable Reactions Can Be Coupled to Favorable Reactions

-a thermodynamically unfavorable (endergonic) reaction can be driven in the forward direction by coupling it to a highly exergonic reaction -often involves coupling to ATP hydrolysis (∆G′° = -30.5 kJ/mol)

Transphosphorylation Examples

-adenylate kinase: lowers the ADP concentration and replenishes ATP during periods of intense demand for ATP; guanylate kinase is a similar enzyme; 2 ADP → ATP + AMP ... ∆G′° = 0 -creatine kinase: uses the phosphocreatine reservoir to replenish ATP at a rapid rate

Galactose Metabolism and Disease

-galactose is the product of lactose hydrolysis; important component in the infant diet -galactokinase uses ATP to phosphorylate galactose at C-1; *see chp 14 slide 59* Conversion of Galactose to Glucose 1-Phosphate: -conversion proceeds through UDP-galactose and UDPglucose intermediates -galactosemia diseases is caused by a genetic defect in enzymes of this pathway; treatment involves carefully controlling dietary galactose *see chp 14 slide 60*

Glucogenic Amino Acids

-glucogenic amino acids are able to undergo net conversion to glucose -intermediates of the citric acid cycle can also undergo oxidation to oxaloacetate *see chp 14 slide 104 for examples*

Gluconeogenesis and Glycolysis Share Several Steps

-gluconeogenesis and glycolysis are NOT identical pathways running in opposite directions -3 glycolysis reactions are essentially irreversible in vivo and cannot be used in gluconeogenesis; must be bypassed with exergonic reactions (the essentially irreversible glycolytic reactions are characterized by a large negative ∆G) *see chp 14 slide 86 and 87 The FIRST Bypass: Conversion of Pyruvate to Phosphoenolpyruvate Requires Two Exergonic Reactions: -pyruvate is transported from the cytosol into mitochondria or generated from alanine within mitochondria by transamination -pyruvate carboxylase is mitochondrial enzyme that converts pyruvate to oxaloacetate; requires the coenzyme biotin - pyruvate + HCO3 - + ATP⟶ oxaloacetate + ADP + Pi --The Role of Biotin in the Pyruvate Carboxylase Reaction: *see chp 14 slide 89* --Phosphoenolpyruvate Carboxykinase: -phosphoenolpyruvate carboxykinase converts oxaloacetate to PEP; requires Mg2+ and GTP; reversible under intracellular condition -oxaloacetate + GTP ⇄ PEP + CO2 + GDP --The Overall Equation for the First Bypass Reaction: -pyruvate + ATP + GTP + HCO3 − ⟶ PEP + ADP + GDP + Pi + CO2... ∆G′° = 0.9 kJ/mol -Note that, en vivo, the actual free energy is strongly negative (−25 kJ/mol) making the reaction effectively irreversible --First Gluconeogenic Steps Travel Through the Mitochondria: *see chp 14 slide 94* --Alternative Paths from Pyruvate to Phosphoenolpyruvate: -when lactate is the glucogenic precursor, a second bypass predominates -oxaloacetate is directly converted to PEP in the mitochondrion by a mitochondrial isozyme of PEP carboxykinase *see chp 14 slide 95* The SECOND and THIRD Bypasses Are Simple Dephosphorylations by Phosphatases -----The SECOND bypass: --fructose 1,6-bisphosphatase (FBPase-1) converts fructose 1,6-bisphosphate to fructose 6-phosphate by hydrolysis of the C-1 phosphate; requires Mg2+; essentially irreversible - fructose 1,6-bisphosphate + H2O⟶ fructose 6-phosphate + Pi... ∆G′° = −16.3 kJ/mol -----The THIRD bypass: --glucose 6-phosphatase = catalyzes the simple hydrolysis of glucose 6-phosphate to glucose; requires Mg2+; only found in the lumen of the endoplasmic reticulum of hepatocytes, renal cells, and epithelial cells of the small intestine -glucose 6-phosphate + H2O⟶ glucose + Pi. ... ∆G′° = −13.8 kJ/mol

Gluconeogenesis

-gluconeogenesis is the pathway that converts pyruvate and related three- and four-carbon compounds to glucose -occurs in all animals, plants, fungi, and microorganisms -mainly occurs in the liver in mammals; can be generated from amino acids -only some plants and fungi can generate glucose from fatty acids *see chp 14 slide 85*

FBPase-1 Is Allosterically Inhibited by AMP

-high [AMP], which corresponds to low ATP, inhibits FBPase; slows glucose synthesis -high [ATP] slows glycolysis and speeds gluconeogenesis *see chp 14 slide 114*

1) Making or breaking C-C Bonds (Homolytic and Heterolytic Cleavage)

-homolytic cleavage: cleavage of a covalent bond where each atom leaves the bond as a radical, carrying one unpaired electron -heterolytic cleavage: cleavage of a covalent bond where one atom retains both bonding electrons; more common

5) Oxidation-Reduction Reactions

-in many biological oxidations, a compound loses two electrons and two hydrogen ions; catalyzed by dehydrogenases -carbon atoms exist in different oxidation states, depending on the elements with which they share electrons: Least oxidized--alkane->alcohol->aldehyde (ketone)->carboxylic acid->carbon dioxide--Most oxidized

lipoate

-lipoate is a coenzyme with two thiol groups that can undergo reversible oxidation to a disulfide bond (-S-S-) -serves as an electron (hydrogen) carrier and an acyl carrier -covalently linked to E2 via a lysine residue *see chp 16 slide 24*

Nucleophiles and Electrophiles

-nucleophiles: functional groups rich in and capable of donating electrons; combine with and give up electrons to electrophiles -electrophiles: electron-deficient functional groups that seek electrons *carbon can act as either a nucleophile or electrophile*

Reactions of the Citric Acid Cycle

-one oxaloacetate molecule can theoretically oxidize an infinite number of acetyl groups because it is regenerated in the cycle -energy from the four oxidations is conserved as NADH and FADH2 *see chp 16 slide 34* The Chemical Logic of the Citric Acid Cycle: -carbonyl groups are more chemically reactive than a methylene group or methane because it is easier to oxidize CO groups -each step of the cycle involves either: an energy-conserving oxidation or placing functional groups in position to facilitate oxidation or oxidative decarboxylation citrate formed from acetyl-CoA and oxaloacetate is oxidized to yield: - CO2 - NADH - FADH2 - GTP or ATP EIGHT STEPS OF THE CITRIC ACID CYCLE: Step 1. Formation of Citrate --citrate synthase catalyzes the condensation of acetyl-CoA with oxaloacetate to form citrate -involves the formation of a transient intermediate, citroyl-CoA -large, negative ∆G′° is needed because [oxaloacetate] is normally very low *see chp 16 slide 39 --Structure of citrate synthase: -binding of oxaloacetate creates a binding site for acetyl-CoA -induced fit decreases the likelihood of premature cleavage of the thioester bond of acetyl-CoA --Mechanism of Citrate Synthase: -Acid/Base Catalysis: *see chp 16 slide 41 and 42* -Thioester Hydrolysis: *see chp 16 slide 43* Step 2. Formation of Isocitrate via Cis-Aconitate: --aconitase (aconitate hydratase) catalyzes the reversible transformation of citrate to isocitrate through the intermediate cisaconitate -addition of H2O to cis-aconitate is stereospecific -low isocitrate concentration is needed to pull the reaction forward since it is thermodynamically unfavorable -we want isocitrate (secondary alcohol) rather than citrate (tertiary alcohol) because secondary alcohols are better substrates for oxidation *see chp 16 slide 44* --Iron-Sulfur Center in Aconitase -iron-sulfur center acts both in the binding of the substrate to the active site and in the catalytic addition or removal of H2O Step 3. Oxidation of Isocitrate to α-Ketoglutarate and CO2: --isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate -Mn2+ interacts with the carbonyl group of the oxalosuccinate and stabilizes the transiently-formed enol -specific isozymes for NADP+ (cytosolic and mitochondrial) or NAD+ (mitochondrial) *see chp 16 slide 46* Step 4. Oxidation of α-Ketoglutarate to Succinyl-CoA and CO2: --α-ketoglutarate dehydrogenase complex = catalyzes the oxidative decarboxylation of α-ketoglutarate to succinylCoA and CO2 -energy of oxidation is conserved in the thioester bond of succinyl-CoA; highly thermodynamically favorable (irreversible) *see chp 16 slide 47* --Note A Conserved Mechanism for Oxidative Decarboxylation: -pathways use the same five cofactors, similar multienzyme complexes, and the same enzymatic mechanism (conserved mechanism) -have homologous E1 and E2 and identical E3 -example of gene duplication and divergent evolution *see chp 16 slide 50* Step 5. Conversion of Succinyl-CoA to Succinate: --succinyl-CoA synthetase (succinic thiokinase) catalyzes the breakage of the thioester bond of succinyl-CoA to form succinate -energy released from breakage of thioester bond drives the synthesis of a phosphoanhydride bond in either GTP or ATP (substrate level phosphorylation) *see chp 16 slide 51* --The Succinyl-CoA Synthetase Reaction: -enzyme molecule becomes phosphorylated at a His residue in the active site -phosphoryl group is then transferred to ADP or GDP to form ATP or GTP (animal cells have specific isozymes for ADP and GDP) -power helices place the partial positive charges of the helix dipole near the phosphate group of the α chain phosphorylated His246 to stabilize the phosphoenzyme intermediate *see chp 16 slide 52 and 53* --Nucleoside Diphosphate Kinase: -nucleoside diphosphate kinase catalyzes the reversible conversion of GTP and ATP in the reaction: GTP + ADP ⇌ GDP + ATP... ∆G′° = 0 kJ/mol -net result of the activity of either isozyme of succinyl-CoA synthetase is the conservation of energy as ATP Step 6. Oxidation of Succinate to Fumarate: --succinate dehydrogenase (also acts a part of complex II in electron transport chain) is a flavoprotein that catalyzes the reversible oxidation of succinate to fumarate (while it is reversible the cycle tends to go in the forward direction by keeping the product concentration low) -integral protein of the mitochondrial inner membrane in eukaryotes -contains three iron-sulfur clusters and covalently bound FAD *see chp 16 slide 57* Step 7. Hydration of Fumarate to Malate: --fumarase catalyzes the reversible hydration of fumarate to L-malate -transition state is a carbanion -slightly thermodynamically favorable *see chp 16 slide 60* --Fumarase Is Highly Stereospecific: -in the forward direction, fumarase catalyzes hydration of the trans double bond of fumarate but not the cis double bond of maleate -in the reverse direction, fumarase is equally stereospecific Step 8. Oxidation of Malate to Oxaloacetate: --L-malate dehydrogenase catalyzes the oxidation of L-malate to oxaloacetate, coupled to the reduction of NAD+ -low oxaloacetate concentration pulls the reaction forward -regenerates oxaloacetate for citrate synthesis *see chp 16 slide 62* The Energy of Oxidations in the Cycle Is Efficiently Conserved: -energy released by oxidation is conserved in the production of: - 3 NADH - 1 FADH2 - 1 GTP (or ATP) *see chp 16 slide 65*

The PDH Complex Catalyzes an Oxidative Decarboxylation

-oxidative decarboxylation is an irreversible oxidation process in which the carboxyl group is removed, forming CO2 *see chp 16 slide 19* -the useful part of a complex is that the regulation of one subunit can affect the entire complex's activity

Which pathway leads to a loss of carbon, which is undesirable in organisms that cannot fix carbon?

-pentose phosphate (oxidative phase) -The 6-phosphogluconate dehydrogenase reaction oxidatively decarboxylates 6-phosphogluconate to form the ketopentose ribulose 5-phosphate

Pentose Phosphate Pathway (PPP)

-pentose phosphate pathway (phosphogluconate pathway, hexose monophosphate pathway) is a pathway that oxidizes glucose 6- phosphate, producing pentose phosphates and NADPH *see image* The Oxidative Phase Produces NADPH and Pentose Phosphates (ribose 5-phosphate): -glucose 6-phosphate dehydrogenase (G6PD) catalyzes the oxidation of glucose 6-phosphate to 6- phosphoglucono-δ-lactone using NADP+ as the electron acceptor *see chp 14 slide 125* Lactonase Hydrolyzes Lactone to 6-Phosphogluconate: -lactonase catalyzes the hydrolysis of lactone to the free acid 6-phosphogluconate *see chp 12 slide 126* 6-Phosphogluconate Dehydrogenase Forms Ribulose 5-Phosphate: -6-phosphogluconate dehydrogenase catalyzes the oxidation and decarboxylation of 6- phosphogluconate to form ribulose 5-phosphate and NADPH *see chp 14 slide 127* Phosphopentose Isomerase Generates Ribose 5-Phosphate: -phosphopentose isomerase converts ribulose 5-phosphate to its aldose isomer, ribose 5- phosphate *see chp 14 slide 128* The Overall Equation for the Pentose Phosphate Pathway: - glucose 6-phosphate + 2NADP+ + H2O ⟶ ribulose 5-phosphate + CO2 + 2NADPH + 2H+

standard transformed constants

-physical constants based on the biochemical standard state Examples: -[H+ ] is 10−7 M -[H2O] is 55.5 M -[Mg2+] is 1 mM -standard free-energy change, ∆G′° -standard equilibrium constant, K′ eq

Glycolysis and Gluconeogenesis Are Reciprocally Regulated

-simultaneous operation of both pathways at each of the three bypass points would consume ATP without accomplishing chemical or biological work! -regulation prevents wasteful operation of both pathways at the same time

Equilibrium Constants Are Multiplicative

-the K′ eq for the overall reaction is the product of the individual K′ eq values for the two reactions -Example: For the phosphorylation of glucose: --Reaction A: K′ eq = ([glucose-6-phosphate]/[glucose] [Pi]) = 3.9 x 10^-3 M --Reaction B: K′ eq = ([ADP] [Pi]/[ATP]) = 2 x 10^5 M --Reaction A coupled with B: K′ eq = ([glucose-6-phosphate] [ADP]/[glucose] [ATP]) = 7.8 x 10^2 M **By coupling reactions, K′ eq has been raised by 2 x 10^5

Electrons from NADH and FADH2 Enter the Respiratory Chain

-the citric acid cycle directly generates only one ATP per turn --the large flow of electrons into the respiratory chain via NADH and FADH2 leads to formation of almost 10 times more ATP during oxidative phosphorylation -each NADH drives formation of ~2.5 ATP -each FADH2 drives formation of ~1.5 ATP

Nonoxidative Reactions of the Pentose Phosphate Pathway:

-the conversion of pentose phosphates to glucose 6- phosphate begins the oxidative cycle again *see image* The Nonoxidative Phase Recycles Pentose Phosphates to Glucose 6-Phosphate: -ribulose 5-phosphate epimerase epimerizes ribulose 5-phosphate to xylulose 5-phosphate *see chp 14 slide 134* -transketolase catalyzes the transfer of a twocarbon fragment from a ketose donor to an aldose acceptor The First Transketolase Reaction: -the first transketolase reaction yields sedoheptulose 7- phosphate *see chp 14 slide 135* The Transaldolase Reaction: -transaldolase catalyzes the condensation of a threecarbon fragment from sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate, forming fructose 6-phosphate and the tetrose erythrose 4-phosphate *see chp 14 slide 136* The Second Transketolase Reaction : -the second transketolase reaction forms fructose 6- phosphate and glyceraldehyde 3-phosphate from erythrose 4-phosphate and xylulose 5-phosphate *see chp 14 slide 137* Stabilization of the Carbanion Intermediates: -thiamine pyrophosphate (TPP) stabilizes a two-carbon carbanion in the transketolase reaction -protonated Schiff base stabilizes the carbanion in the transaldolase reaction The Oxidative and Nonoxidative Reactions of the Pentose Phosphate Pathway Reversability: -the first and third steps of the oxidative pentose phosphate pathway are essentially irreversible in the cell -the nonoxidative reactions are readily reversible

The Free-Energy Change for ATP Hydrolysis Is Large and Negative

-the hydrolytic cleavage of the terminal phosphoanhydride bond in ATP relieves some of the internal electrostatic repulsion in ATP -There is resonance stabilization of the leaving group *see chp 13 slide 33 for image*

Transphosphorylations between Nucleotides Occur in All Cell Types

-the other nucleoside triphosphates (GTP, UTP, and CTP) and all deoxynucleoside triphosphates (dATP, dGTP, dTTP, and dCTP) are energetically equivalent to ATP -nucleoside diphosphate kinase = carries phosphoryl groups from ATP to other nucleotides; this reaction happens when there is a high [ATP]/[ADP] ratio

Glucose 6-Phosphate Is Partitioned between Glycolysis and the Pentose Phosphate Pathway

-the relative concentrations of NADP+ and NADPH determine whether glucose 6-phosphate enters glycolysis or the pentose phosphate pathway *see chp 14 slide 140*

Gluconeogenesis Is Energetically Expensive, But Essential

-the sum of the biosynthetic reactions leading from pyruvate to free blood glucose is: 2 pyruvate + 4ATP + 2GTP + 2NADH + 2H+ + 4H2O ⟶ glucose + 4ADP + 2GDP + 6Pi + 2NAD+ -Essential because the brain, nervous system, and red blood cells can only generate ATP from glucose -Gluconeogenesis is especially important during nutrient starvation or vigorous exercise because we tend to be low on glycogen then

4) Group Transfer Reactions

-the transfer of protons, acyl, glycosyl, and phosphoryl groups from one nucleophile to another is common -acyl group transfer involves the addition of a nucleophile to the carbonyl carbon of an acyl group to form a tetrahedral intermediate Phosphoryl Group (usually ATP) Transfers: -attachment of a good leaving group to a metabolic intermediate "activates" the intermediate for subsequent reaction -phosphoryl groups (-PO3 2- ) commonly serve as leaving groups -kinases are enzymes that catalyze phosphoryl group transfers with ATP as a donor

Thiamine Pyrophosphate (TPP)

-thiamine pyrophosphate is the coenzyme derived from vitamin B1 -the thiazolium ring plays an important role in the cleavage of bonds adjacent to a carbonyl group *see slide 74 and 75 for examples*

Three Catabolic Fates of Pyruvate

-under aerobic conditions, pyruvate is oxidized to ACETYL-COA -under anaerobic conditions or low oxygen condition (hypoxia), pyruvate is reduced to LACTATE or ETHANOL -NADH must be recycled to regenerate NAD or else will be inhibited *see chp 14 slide 68* Pyruvate Is the Terminal Electron Acceptor in Lactic Acid Fermentation: -organisms can regenerate NAD+ by transferring electrons from NADH to pyruvate, forming lactate -lactate dehydrogenase catalyzes the reduction of pyruvate to LACTATE *see chp 14 slide 69* --Reduction of Pyruvate to Lactate Regenerates NAD+ so glycolysis can continue to occur -glycolysis converts 2NAD+ to 2NADH -reduction of pyruvate to lactate regenerates 2NAD+ -there is no net change in NAD+ or NADH *see chp 14 slide 70* ETHANOL Is the Reduced Product in Ethanol Fermentation: -yeast and other microorganisms regenerate NAD+ by reducing pyruvate to ethanol and CO2 -the overall equation is: glucose + 2ADP + 2Pi ⟶ 2 ethanol + 2CO2 + 2ATP + 2H2O *see chp 14 slide 71*

Laws of Thermodynamics; all organisms follow these rules

1) energy can not be created or destroyed, only converted from one form to another 2) the universe always tends toward increasing disorder/entropy (however cells like to be structured so they tend to combat this)

Five general categories of reactions in living cells:

1) reactions that make or break carbon-carbon bonds 2) internal rearrangements, isomerizations, and eliminations 3) free-radical reactions 4) group transfers 5) oxidation-reductions

Five General Principles of Metabolic Pathways

1. Complete metabolic pathways are irreversible 2. Catabolic and Anabolic Pathways must differ 3. Every metabolic pathways has a committed step 4. All metabolic pathways are heavily regulated 5. Metabolic pathways in eukaryotic cells are located in specific compartment

Endergonic

A chemical reaction that requires the input of energy in order to proceed.

Chapter 13 Principle 3

Although thousands of different chemical reactions occur in the biosphere, most of them fall within a small set of reaction types.

ATP donates Phosphoryl groups (but also Pyrophosphoryl, and Adenylyl Groups)

each of the three phosphates of ATP is susceptible to nucleophilic attack *see chp 13 slide 38*

Energy Remaining in Pyruvate

energy stored in pyruvate can be extracted by: --aerobic processes: -oxidative reactions in the citric acid cycle -oxidative phosphorylation --anaerobic processes: -reduction to lactate -reduction to ethanol -pyruvate can provide the carbon skeleton for alanine synthesis or fatty acid synthesis

ATP hydrolysis is generally written as one step reaction to keep things simple but it is actually 2 steps...

first step = transfer of part of the ATP molecule to a substrate molecule or to an amino acid residue, activating it second step = displacement of the phosphate-containing moiety, generating Pi , PPi , or AMP as the leaving group *chp 13 slide 37*

Reduction

gain of electrons

3) Free-Radical Reactions

homolytic cleavage of covalent bonds to generate free radicals occurs in some pathways

Oxidation

loss of electrons

Exergonic

reactions that release energy

Metabolism

sum of all chemical reactions in an organism

The PDH Complex Enzymes

the PDH complex contains multiple copies of: - pyruvate dehydrogenase (E1 ) - dihydrolipoyl transacetylase (E2 ) - dihydrolipoyl dehydrogenase (E3 ) -an E2 core (of 24-60 copies) is surrounded by multiple and variable numbers of E1 and E3 copies

Bioenergetics

the Quantitative Study of Energy Transductions

The Relationship Between K′ eq and ∆G′°

∆G′° = -RT ln K′ eq -standard free-energy change, ∆G′° -standard equilibrium constant, K′ eq -Temperature, T -R is ..?

Glucose

Central Importance of Glucose: -Glucose is an excellent fuel. -Glucose is a versatile biochemical precursor. Major Pathways of Glucose Utilization: *see chp 14 slide 4*

The Citric Acid Cycle Serves in Both Catabolic and Anabolic Processes

-amphibolic pathway is one that serves in both catabolic and anabolic processes -animals cannot convert acetate or acetyl-CoA to glucose; in the citric acid cycle, there is no net conversion of acetate to oxaloacetate -glyoxylate cycle is a reaction sequence that converts acetate to carbohydrate; present in bacteria, plants, fungi, and protists Role of the Citric Acid Cycle in Anabolism: *see chp 16 slide 75* Anaplerotic Reactions Replenish Citric Acid Cycle Intermediates: -when intermediates are shunted from the citric acid cycle to other pathways, they are replenished -anaplerotic reactions are chemical reactions that replenish intermediates *see examples in chp 16 alide 76*

Mammals Cannot Convert Fatty Acids to Glucose; Plants and Microorganisms Can

-animals lack the enzymatic machinery to convert acetylCoA from fatty acids to pyruvate. -plants and microorganisms have the glyoxylate pathway, which allows them to make glucose from fatty acids

Anabolism

-biosynthesis, uses energy -the building phase of metabolism; requires energy

Transcriptional Regulation Changes the Number of Enzyme Molecules

-by regulating enzymes you can regulate the pathways that the enzymes promote -insulin transcriptionally regulates more than 150 genes *see chp 14 slide 121*

Cellular Respiration

-cellular respiration is the process by which the pyruvate produced by glycolysis is further oxidized to H2O and CO2 *see chp 16 slide 3* -process by which cells consume O2 to produce H20 and CO2 -provides most of the ATP in the cells through various catabolic pathways (moreso than glycolysis) -can also capture energy stored in lipids and amino acids --Stage 1 of Cellular Respiration: -Oxidation of fuels to acetyl-CoA -generates ATP, NADH, FADH2 *see chp 16 slide 4* Stage 2 of Cellular Respiration: -oxidation of acetyl groups to CO2 in the citric acid cycle (tricarboxylic acid (TCA) cycle, Krebs cycle) -nearly universal pathway -generates NADH, FADH2 , and one GTP *see chp 16 slide 5* Stage 3 of Cellular Respiration: -electron transfer chain and oxidative phosphorylation -generates the vast majority of ATP from catabolism *see chp 16 slide 6* -electrons funnels through the respiratory chain (in plasma membrane of bacteria); reduces oxygen to form water and the drive of electrons produce ATP In animals: -Glycolysis mostly occurs in the cytoplasm -Citric acid cycle occurs in the mitochondria (mitochondrial matrix and one reaction in the inner membrane of the mitochondria) -Oxidative phosphorylation occurs in the inner membrane of the mitochondria

Fermentations Produce Some Common Foods and Industrial Chemicals

-certain microorganisms in food products ferment the carbohydrates and yield metabolic products that give the foods their characteristic forms, textures, and tastes -the drop in pH preserves food from spoilage Fermented Foods: -Lactobacillus bulgaricus ferments carbohydrates in milk to lactic acid to make yogurt -Propionibacterium freudenreichii ferments milk to produce propionic acid and CO2 to make Swiss cheese -other examples: pickles, sauerkraut, sausage, soy sauce, kimchi, kefir, dahi, and kombucha Fermented Beverages: -ethanol fermentation of carbohydrates in cereal grains by yeast glycolytic enzymes produces beer

Allosteric Regulation of PFK-1 By Citrate

-citrate is a key intermediate in the aerobic oxidation of pyruvate, fatty acids, and amino acids -citrate allosterically regulates PFK-1; high concentrations increase the inhibitory effect of ATP; serves as an intracellular signal that the cell is meeting its current needs for energy-yielding metabolism by the oxidation of fats and proteins

Coenzyme A (CoA-SH)

-coenzyme A has a reactive thiol (-SH) group that is critical to its role as an acyl carrier -the -SH group forms a thioester with acetate in acetyl-CoA *see structure in chp 16 slide 14* -role is to accept acyl groups, carry acyl groups (particular acetyl groups)

Catabolism

-energy producing -the degradative phase of metabolism; releases energy

Pyruvate Is Oxidized to Acetyl-CoA and CO2

-enters outer mitochondrial membrane via via facilitated diffusion -mitochondrial pyruvate carrier (MPC) is an H+ -coupled pyruvate-specific symporter in the inner mitochondrial membrane -pyruvate dehydrogenase (PDH) complex is a highly ordered cluster of enzymes and cofactors that oxidizes pyruvate in the mitochondrial matrix to acetyl-CoA and CO2; the series of chemical intermediates remain bound to the enzyme subunits; regulation results in precisely regulated flux

Free-Energy Changes and Enzymes

-enzymes cannot change equilibrium constants however they can increase the rate at which a reaction proceeds -the free-energy change for a reaction is independent of the pathway by which the reaction occurs -Energetics within a cell are non-standard: --Depends on ∆G′° --Depends on actual concentrations of products and reactants

Ping-Pong Mechanism of Nucleoside Diphosphate Kinase

-first step: phosphoryl group transfer from ATP to an active-site His residue produces a phosphoenzyme intermediate -second step: transfer of the phosphoryl group is from the His residue to an NDP acceptor *see chp 13 slide 40*

Standard Free-Energy Changes Are Additive

-for two sequential chemical reactions, A ⇄ B and B ⇄ C, each chemical reaction has its own equilibrium constant and standard free-energy change ∆G1 ′° and ∆G2 ′° -for the overall reaction, A ⇄ C, the standard free-energy change is the sum of the individual standard free-energy changes

Fructose 2,6-Bisphosphate Is a Potent Allosteric Regulator of PFK-1 and FBPase-1

-fructose 2,6- bisphosphate mediates the rapid hormonal regulation of glycolysis and gluconeogenesis -binds to PFK-1 and increases its affinity for fructose 6- phosphate -binds to FBPase-1 and reduces its affinity for its substrate

It is uncommon for one molecule to act as both an activator and inhibitor in metabolism. Which molecule both activates glycolysis and inhibits gluconeogenesis? A. NAD+ B. ADP C. pyruvate D. fructose 2,6-bisphosphate E. glucose 6-phosphate

-fructose 2,6-bisphosphate -Fructose 2,6-bisphosphate (F26BP) activates PFK-1, stimulating glycolysis. At the same time, F26BP inactivates FBPase-1, inhibiting gluconeogenesis

Chapter 14 Principle 3

Pyruvate formed under anaerobic conditions is reduced to lactate with electrons from NADH, recycling NADH to NAD+ and allowing continued glycolysis in the processes of lactate or alcohol fermentation

Hexokinase Isozymes

Hexokinase Isozymes Are Affected Differently by Their Product (Glucose 6- Phosphate): -hexokinase I, hexokinase II, and hexokinase III are all inhibited by their product, glucose 6-phosphate -hexokinase IV (glucokinase) in the liver is not inhibited by glucose 6-phosphate due to its kinetic properties Kinetic Properties of Hexokinase IV and Hexokinase I: -hexokinase IV (glucokinase) in the liver has kinetic properties related to its role in maintaining glucose homeostasis -Km (binding affinity) is higher than the usual glucose concentration *see chp 14 slide 110* Regulation of Hexokinase IV by Sequestration in the Nucleus: -the regulatory protein anchors hexokinase IV inside the nucleus, where it is segregated from the other glycolytic enzymes -fructose 6-phosphate is an allosteric effector -glucose causes dissociation of the regulatory protein *see chp 14 slide 111*

Hexokinase

Hexokinase is Present in Nearly All Organisms -humans encode 4 hexokinases (I to IV) that catalyze the same reaction -Hexokinase one-four are examples of isozymes (two or more enzymes that catalyze the same reaction but are encoded by different genes) -hexokinase is used in step 1 of glycolysis

Only a Small Amount of Energy Available in Glucose Is Captured in Glycolysis:

In total it would be delta ∆G′° = -2,840 kJ/mol *see chp 16 slide 2*

Chapter 14 Principle 1

Metabolites like glucose are often activated with a high-energy group before their catabolism.

The PDH Complex Channels Its Intermediates through Five Reactions

PDH 5 intermediates: *reactions in chp 16 slide 28* step 1 is the rate limiting step The Five-Reaction Sequence of the PDH Complex Is An Example of Substrate Channeling: -substrate channeling is the passage of intermediates from one enzyme directly to another enzyme without release of those intermediates - this is made possible by the long lipoyllysyl arm of E2. It channels the substrate from the active site of E1 to E2 to E3: 1) tethers intermediates to the enzyme complex, 2) increases the efficiency of the overall reaction, 3) and minimizes side reactions

The PDH Complex Employs Three Enzymes and Five Coenzymes to Oxidize Pyruvate

PDH is composed of three enzymes: - pyruvate dehydrogenase, E1 - dihydrolipoyl transacetylase, E2 - dihydrolipoyl dehydrogenase, E3 PDH has five coenzymes: - thiamine pyrophosphate (TPP) - lipoate - coenzyme A/pantothenate (CoA, CoA-SH) - flavin adenine dinucleotide/riboflavin (FAD) - nicotinamide adenine dinucleotide/niacin (NAD)

anaerobic

Process that does not require oxygen

aerobic

Process that requires oxygen

energy transductions

changes of one form of energy into another