Exam 4 MCDB 310 Akey

1. NADH DH 2. Succinate dehydrogenase- complex II 3. Ubiquninon:cytochrome c oxidoreductase-- Q Cycle 4. Cytochrome oxidase-- complex IV now the actual steps for complex 4, no iron/s but copper instead Balance sheet: used in each complex and number protons

- 4 e- used to reduce an O2 molecule into 2 H2O molecules (2 for .5 O2 or one oxygen) - Four protons are picked up from the matrix in the process and passed to the IMS (C1 = 4 protons, C2 = 0, C3 = 4, C4 = 2) Cytochrome C carry in one electron each and pass to complex IV to reduce 1/2 O2. - 2 complete Q cycles needed to reduce O2 to 2 H2O Send 2 H to IMS from 1/2 O2! uses iron-copper center Copper center hemes Balance Sheet: C1: 4 H+ to IMS with NADH and QH2 C2: 0 H+ to IMS with FADH2 and QH2 CIII: QH2 Q cycle pumps 4 H+ out, make 2 cytochromec CIV: 2 H+ out per 1/2 O2, use cyt c

Before Fatty acid modification... now Cholesterol Synthesis START! main notes - all can be from (simple precusors) - X carbons - essential in diet? - key intermediate Cholesterol synthesis 4 stages --------- in depth on stage 1: acetate -> mevalonate A+B found where else A. B. C. (com step, why good?)

- All can be made from acetyl-CoA (acetate is precursor) - 27 carbons - Essential in cell membranes but can be synthesized, not essential in diet - Isoprene is key intermediate! 40 total steps 1. Synthesis of mevalonate (6-carbon) from 3 acetates (2 carbon) 2. 6 Mevalonate converted to 6 activated isoprenes (5-carbon) 3. Condensation of 6 isoprenes to form squalene ->Conversion of squalene into four ring steroid nucleus Lanosterol 4. Lanosterol has 20 more steps to modify it to cholesterol ------------------ Stage 1: Acetate -> Mavelonate A. 2 acetyl-CoA -> Acetoacetyl-CoA Thiolase B. AcetoAcetyl-CoA + Acetyl-CoA -> HMG-CoA HMG-CoA Synthase C. HMG-CoA + 2 NADPH + 2 H+ -> Mavelonate + CoA + 2 NADP+ HMG-CoA Reductase ... The committing step toward cholesterol synthesis A + B = reversible!!! Same mechanism of keton body production C = IRREVERSIBLE!!! Specific to cholesterol synthesis, good point for regulation!

Dinitrogenase complex keys: - di of di - similarity with rubisco prosthetic groups quick mechanism ATP cost -------- symbiotic relationship, how bacteria afford ATP debt to do nitrogen fixing

- Dimer of a dimer (alpha/beta dimer come together with another set and form a dimer in a dimer) - Similar with rubisco: Dinitrogenase also has an affinity for O2... O2 will deactivate the complex reapidly (30 seconds) and so this must be done in ANAEROBIC conditions! N needs 4 H+, so each N in N2 needs 4-, 8 e- total Dinitrogenase is reduced by dihydrogenase reducatse ONE electron at a time... Prosthetic groups are: - 4 Fe-S centers - FeMo cofactor (molybdenum) Mechanism: 1. two Dinitrogenase reductase get an e- and deliver 2 e-, both one at a time, to the dimer-dimer reductase complex 2. each transfer requires binding between the complexes Each electron of the 8 transferred, 2 ATP consumed (16 ATP total consumed in the process) ______________________ Bacteria in root nodule of Legumes: - Have Leghemoglobins which bind O2 and prevent corruption of catalyst in dinitrogenase complex. - Bacteria have access to plants energy stores in order to make tons of NH3, excess put into soil!

The ultimate fates of nitrogen in organisms: 1. plant 2. aqua 3. terres 4. desert most energy has alr been... Sum of three products -------- Intermediate nitrogen carriers...ch 18 slide 7

- Plants conserve almost ALL nitrogen, it is usually a limiting element of plant growth since they do not intake it with "diet" unless its in soil - Aquatic vertebrates release ammonia to their environment, passive diffusion from epithelial cells or actively transport it through gills - Terrestrial vertebrates and sharks excrete nitrogen in the form of Urea which is less toxic than ammonia and is VERY SOLUBLE - Some desert animals like birds and reptiles excrete nitrogen as Uric Acid which is NONPOLAR and INSOLUBLE, so it is excreted as a paste allowing these animals to conserve water excretion. In the urea form, most energy has already been exctracted! NH4+: fish and bony fish ... ammonotelic animals Urea: Sharks and terrestrial vertebrates like humans and apes ... ureotelic animals Uric acid: birds, reptiles ... uricotelic animals ---------------------------------------- Pyruvate -> Alanine (Glucose alanine cycle) alpha-ketoglutarate -> glutamate (transaminations) Glutamine <-> Glutamate (Nitrogen from tissues) Oxaloacetate -> Aspartate (Aspartate shunt) Alanine, glutamate, and aspartate all same chemistry of =O replaced with amine group

ETC coupled to ATP synthesis and what stops it graphs: 1. cyanide 2. oligomycin

1. ATP synthesis requires electron transport 2. Electron transport also requires ATP synthesis Oxygen consumed (black) = number electrons going through ETC to create H+ gradient ATP synthesis (red) = movement of motor and translocation of protons Cyanide (CN-) blocks transfer between cytochrome oxidase and O2 - Add in ADP and Pi, add succinate which provides FADH2 electrons and, when you add CN- the oxygen consumption stops and is blocked, this also stops the ATP synthase and proton transport since no proton gradient is established. Oligomycin or Venturicidin: Fo in ATP synthase is oligomycin sensitive. Add this in and the ATP synthase will stop operating and generating ATP and the ETC will stop creating a proton gradient. - HOWEVER: Add a DNP and this process is Uncoupled, the ATP synthase and ATP generation stops, the ETC and Oxygen consumption continues. Uncoupling: ATP synthase is bypassed by O2 consumption, the proton gradient continues to form while no ATP is made.

Four steps of Chain Lengthening with FAS-I 1. cond 2. reduc 3. dehyd - note on cis/tra 4. red

1. Acetyl + Malonyl -> CO2 + AcetoAacetyl-ACP - Condesnation of acetyl + Malonyl group to form Acetoacetyl-ACP ... is bound to ACP's Pantothenate! - Catalyzec by KS (Ketoacyl synthase) - Release CO2 2. Acetoacetyl-ACP + NADPH -> Beta-Hydroxybutyrl-ACP + NADP+ + - Acetoacetyl-CoA will be reduced using NADPH to get rid of beta keto acid - Carbonyl reduction catalyzed by KR (ketoreductase) 3. Beta-Hydroxybutyrl-ACP -> H2O + trans-delta-Butenoyl-ACP - Dehydration of the hydroxy group, forms a double bond! Removal H from C2 and OH from C3 - NOTE: All natural occuring double bonds are CIS!!! This one forming is trans! - Catalyzed by Dehydratase (DH) 4. Butenoyl-ACP + NADPH + H+ -> NADP+ + Butryryl-ACP - reduce double bond using enzyme EnoylReductase (ER) -----> Butyryl-ACP is transferred from ACP to cys on KS, where a new malonyl group attaches ACP, and will attach the KS intermediate to being the C1 of the new growing acyl! ---> Now you have 4 carbon chain! Repeats 6 more times (4 + 12 = 16) and you get plamitoyl-ACP! - release this product from ACP using hydrolysis (requires one water)

Adding polar head group to Diacylglycerol!!! Slide ch21, 41 shows triacylglercol enzymes/formation THREE strategies for adding polar head group... makes -phosphatidyl x3 strategies 1/2: 2 steps to reaction, what is left, so what is energy equivalent used? strat 3: example w ethano-serine Option A Option B

1. Activate the 1,2-Diacylglycerol via CTP (citylylation) 2. Activate polar head group via CTP 3. Head group exchange via one of MANY salvaged pathways Makes: - phosphatidylcholine - phosphatidylserine - phosphatidylethanolamine and more For strategy 1/2 1. Add a CDP to head group of Diacylglycerol or polar head 2. Nucleophilic attack by molecule not with CDP, CMP will leave and a single phosphate remains!!! 1 CTP -> 1 CMP = 2 ATP equivalents per diacylglycerol rxn For Strategy 3: OPTION A: Head groups switch, no energy cost Phosphatidyletehanolamine-serine transferase enzyme switches head groups on and off OPTION B: next slide, modify headgroups to result in diff compound/cul if levels are low and no switch can occur

Review/How we hold on to and transfer biological amines! 1. aminotransferase How to get free ammonia into useable forms... (net increase?) 2. gln - store 3. Glu - net - used by

1. Aminotransferase: Amino acid + keto acid <-> keto acid + amino acid - no net increase in amino acids - a REDOX reaction! - keto acid is reduced and aminoated to form amino acid 2. NH4+ + Glu + ATP -> [glutamyl-phosphate] -> Gln + ADP + Pi + H+ glutamine synthetase --- found in ALL organisms, NO net increase in amino acids! ------ get free ammonia into amino carrier! (less toxic and useable now for other reactions or travel) 3. alpha-ketoglutarate + NADPH + H+ + Gln -> 2 glu + NADP+ Glutamate synthetase - alpha-ketoglut -> glutamate and glutamine donating amido group turns into the second glutamate! - alpha-ketoglutarate is reduced and animated using electrons from NADPH - used by plants and bacteria, NOT present in animals ----- used to net increase amount of amino acids!

End on a new mini topic: Single carbon transfer agents (very important in amino acid metabolism) each carries overview 1 bio - where bio appeared before, what activates it 2 3

1. Biotin: CO2 Carrier, seen in bypass 1 gluconeogenesis - two rings, a valerate side chain with a carboxylic acid group - uses ATP to activate a CO2 and transfer it to biotin - Acetyl-CoA carboxylase prosthetic group! 2. THF - tetrahydrofolate: Most versatile, can carry methyl (-CH3), Methy-hydroxyl (-CH2-OH), and Carbonyl carbon (-CHO) 3. S-Adenosylmethionine (AdoMet): best CH3 transferer

Regulation of the Urea Cycle 1. carbom synt step regulated by.... - this enzyme is activated by, and the step to make it is... - what activates this enz and why does that make sense? 2. Overall express when need trick on what enters the cycle, (CAC and urea)

1. Carbamoyl-phosphate synthase I (take bicarbonate and 2 ATP to form this ammonia carrier) is ACTIVATED by N-acetylglutamate N-acetylglutamate: - A molecule formed solely to regulate the urea cycle - Is made by N-acetylglutamate synthase! - N-acetylglutamate synthase activated by arginine and high glutamate/acetyl Co-A Why does this allosteric activation make sense? - High glutamate (nitrogen carrier) and high arginine (urea cycle intermediate) concentrations are indicators of active amino acid metabolism! So get the Urea cycle going! 2. Expression of urea cycle enzyme increases when needed! - high-protein diet, need lots of processing enzymes - Starvation! when protein/amino acids are being broken down for energy! Pyruvate does not enter CAC, acetyl coa does. :: Ammonia does not enter urea cycle, carbamoyl-phosphate does

Rubisco-- Carboxylase mechanism 1. form enedi 2. polarize w Mg for nucleoph att 3. beta keto + hdyrox 4. cleavage 5. protonate carban KEY NOTE: how much energy is required?

1. Carbomylated Lys attracts Mg2+ which binds rib 1,5BP and forms a enediolate at the active site. 2. Mg next recruits a CO2 which due to charge, it polarizes the CO2 (makes it positive) which faciliates the Enediolate intermediate to nucleophilically attack the CO2, generating a 6-carbon sugar! 3. A beta keto acid intermediate forms, hydroxylation (adding water) occurs at the C3 carbonyl... beta-ketos are very unstable and so it quickly does step 4 4. A hydrated intermediate forms but is cleaved to produce a 3-phosphoglcyerate that leaves, and a 3-carbon complex remains. 5. The remaining 3-carbon is a carbanion (negative carbon) which is protonated by a lysine residue in the active site which generates a second molecule of 3-phosphoglycerate So long as the rubisco is active (rubsico activase, light triggered and ATP used) then NO ENERGY is required, this reaction occurs without any energy! CARBON TRACKING: #1 carbon on second 3-phosphoglycerate formed - first 3-phosphoglycerate formed is all from ribulose

3 key Amino acid regulation strategies! Feedback is

1. Concerted inhibition: Feedback inhibition can be additive or synergistic (sum total = more than the parts)! - example is all of the different negative regulators of glutamine synthetase 2. Enzyme multiplicity: Some steps are catalyzed by several isozymes all with independent modulators... One reaction has a few enzymes to carry our same process but they are regulated differently! Prevents one biosynthetic product from shutting down key steps in a pathway when other downstream products are still required. - In image, aspartate has A1, A2, and A3 enzymes all doing the same reaction!!! - reduce overall pool of enzyme, not stopping all! 3. Sequential inhibition: Some pathways have inhibition at several steps! - threonine regulates multiple steps in its pathway!

malate-aspartate shuttle return 1. used for energy equiv 2. fumarate enter mal/asp shuttle a. mal form oxalo b. oxalo + glut, or Oxalo to TCA Glutamate and two fates of nitrogen: 1. release ammon (earlier this lec) 2. shuttle use to form this other aa...

1. Deliver energy equivalents of NADH to ETC 2. Fumarate from urea cycle can use this shunt to enter mitochondria as malate using cytosolic fumarase! a. Malate enters mitochondria and is oxidized to form an NADH and oxaloacetate! b. Oxaloacetate can be combined with glutamate (enzyme apsartate aminotransferase) to form aspartate. I. aspartate now can go back into cytosol and be used in Urea cycle! OR II. oxaloacetate instead enter citric acid cycle! NOTE ON GLUTAMATE two fates of its nitrogen: 1. It can release ammonia using transdeamination where NH4+ goes to synthesize Urea... or 2. Glutamate can be used in the malate-aspartate shuttle to form aspartate from a aminotransferase to oxaloacetate Aspartate-malate shuttle is a key provider of aspartate for the urea cycle!

ATP Synthase functional domains: Fo: function, o stands for F1: function, drxn, subunits

1. Fo: an integral membrane protein with a proton pore (oligomycin-sensitive "O" in Fo, it will block ATP synthe.) - transports protons from IMS to matrix dispiating the proton gradient - a transmembrane protein, has C subunit which comporise the rotor .. c = carousel - a subunit forms the channel protons transport through 2. F1: Peripheral membrane protein that in isolation will solely hydrolyze ATP, it is an ATPase which can convert ADP + Pi to ATP or it can go ATP -> ADP... all enzymes are REVERSIBLE... it is dependent upon Fo direction of H+ transport Nine Subunits: 5 different kinds - alpha3 and beta3: form a heterohexamer, or 3 dimers of alpha/beta around the central stalk. Beta subunits have the catalytic sites. This domain is in the Matrix, N side - B2 = the lever - gamma: The stalk subunit holds handle and rotors together

Lecture 23 Chapter 21-- Lipid Anabolism overview of glycerophospholipid synthesis! (make, lenth, desat, add glyc, head) Catabolism vs anabolism: 1. req vs prod 2. produce vs cons 3. takes place where -- note on paths

1. Make 16 carbon fully saturated fatty acid chains (palmitic acid product) -- the main one for mammals 2. Lengthen acyl chain if necessary 3. Desaturate acyl chain if necessary 4. Add glycerol backbone if necessary 5. Add head group to glycerol backbone if necessary Anabolism: 1. requires Acetyl-CoA (2C) and Malonyl-CoA (3C) 2. Requires reducing power from TWO NADPH 3. Takes place in the CYTOSOL in animals, Chloroplast in plants Catabolism: 1. Produces Acetyl-CoA (2C) 2. Produces reducing power NADH and FADH2 3. Occurs in the mitochondria! --NOTE: Anabolism and catabolism both use different enzymes! Pathways must have diff enzymes based on ch 3 all reaction guidelines!

Goal 1: Collecting electrons, and attaching to membrane bound carriers... -> Mitochondrial Structure 4 parts 1. O spac: holes, permeability 2. ims high of 3. I : what is here, permeability, gradient, SA with ? 4. Mat: location where what happens, concentr is ___ here IMAGE SHOWS what enzymes each component contains!

1. Mitochondrial outer membrane - POROUS, allow passage of metabolites across - freely permeable to ions and small solutes 2. Inner membrane space (IMS) - Similar to cytosol enviornment - HIGH concentration of H+ (called P-side) 3. Mitochondrial Inner membrane - Relatively IMPERMEABLE! Has a proton gradient across it - ETC is situated here - Convolutions are called CRISTAE, increase surface area for more oxidative phosphorylation to take place 4. mitochondrial matrix - Citric acid cycle occurs here, plus parts lipid and amino acid metabolism - LOW proton concentration -- the N side

Overview steps in FA synthesis

1. ONLY the first step does an acetyl group Bind the fatty acid synthase enzyme and so does a malonyl group.. Condensation reaction occurs and the malonyl loses a CO2 and attacks the Acetyl group. 2. The Beta-acid component of the malonyl group attached to acetyl will be REDUCED using NADPH (forming NADP+) 3. Dehydration occurs forming a double bond 4. Reduction occurs again and the fatty acid acyl group remains now 2 carbons longer! Oxidation of FA: Oxid-hydrate-oxidize, now its reverse!

Overview of Catabolism of amino acids: 1. get aa 2. break into x and y 3. a. X can produce ... b. Y can produce d OR e

1. Obtain amino acids either through diet or from oxidation of a protein. 2. Produce amino acids which can break down into ammonium and carbon skeleton 3. a. Carbon skeleton will be converted to alpha-keto acids and can produce energy in krebs cycle b. the ammonia will be converted to biosynthesis of amino acids, nucleotides, and amines OR will be transferred as carbamoyl phosphate to the urea cycle to be excreted from the body The urea cycle and citric acid cycle are interconnected!

Photosynthesis BIG PICTURE: a. photo b. fix ------- CO2 assimil - animals/ plants use... - gluconeogenesis/glycolysis diff? regulation types for animals vs plants...

1. Photophosphorylation (thylakoid membrane) -> oxidize H2O to produce O2, ATP, and NADPH thorugh electron transfer/exciton reactions 2. Carbon Fixation (stroma)-> Reduce CO2 from the environment to synthesize (anabolism) sugars, amino acids, and fatty acids note: both occur in chloroplast! ----------------------- Both animals and plants use 3-carbon intermediates for synthesis... but plants can MAKE the 3-c intermediate Animals = pyruvste, lactate, etc. Plants= make Glyceraldehyde 3-Phosphate using photosynthesis NADPH and ATP Plants and animals both due gluconeogenesis/glycolysis IDENTICALLY Plants and animals both use allosteric effectors and covalent modification for regulation! PLANTS also... - reduce cys-cys crosslinks using electron transporting in PSI - Change pH and [Mg2+] from illumination

Chlorplast anatomy... light energy converted to ATP overview mechanism - orientation ATPase

1. Stroma: the inner lumen of the thylakoid, nearly identical to the mitochondrial matrix make up 2. Thylakoids: Complex membrane systems where photphosphorylation occurs, form stacks, similar in function about surface area to cristae in mitochondria a. Stromal thylakoids: flattened highway like portions b. Granal thylakoids (grana): stacks ------- Light energy -> ATP 1. Light converts H2O to a good e- donor, feeding electrons to thylakoid membrane 2. thylakoid membrane pumps H+ in as electrons flow to NADP+, the flow also generates energy stored as electrochemical potential 3. Energy of H+ potential on P-Side (Thylakoid lumen side) used to make ATP in the stroma orientation: F1 in stroma, Fo in thylakoid membrane

Now we got here to the liver either from glutamine (tissues) or alanine (muscles), what catabolism do we do now in the liver? three general classes (energy fav/netru/cost for each?) 1. transaminations: - catalyzed by - transfer ... generate... - cofactor? 2. Oxida deam - catalyzed by - remove, form, and produce 3. Glutaminase (just covered a couple slides back!) - catalyzed by - gln stores - purpose?

1. Transaminaitons: energetically neutral reaction - catalyzed by aminotransferases, each amino acid has its own enzyme - Transfer amine groups from one amino acid typically to an alpha-ketoglutarate - produce a glutamate and a alpha-keto acid - Uses the pyridoxal phosphate cofactor 2. Oxidative deamination: energetically favorable - Catalyzed by glutamate dehydorgenase - Remove an amino group from glutamate and form alpha-ketoglutarate, release free ammonia likely to be used to form carbamoyl phosphate! 3. Glutaminase: Energetically unfavorable, req 1 ATP - Catalyzed by glutaminase - glutamine is a temporary storage unit of nitrogen (transfers from tissues to blood to liver) - Glutamine can donate an ammonia for aa biosynthesis 3 amino group carriers: Glutamine= carry 2 aminos from tissues to liver, gives up first free N to make carbamoyl phosphate Glutamate= 3 paths, but can aminotransferase with oxaloaccetate to form aspartate, deliver N to urea alanine= muscles to liver

Coupling proton translocation to ATP synthesis CRYO-EM (slide 52 ch 19)... - 1/2 ch mechanism transporting protons

1. proton translocation through a channels causes a rotation of the Fo subunit and central shaft gamma 2. this causes a conformational change within all the three alpha/beta pairs 3. The conformational change in one of the three pairs promotes condensation of ADP and Pi into ATP Transport of protons shown by CRYO-EM: 1. a subunit is the proton half-channel. Called this becuase protons only go halfway 2. At the halfway mark, negative aspartate residues on the c (carousel) subunit binds the positive proton 3. This makes it neutrally charged and so now the carousel can spin around into the intermembrane space 4. It will spin until it reaches the positively charged ARGININE residue which repels and kicks off protons into the second half channel where it enters the mitochondrial matrix

How does H2O end up being oxidized and donating an electron when it is so unfavorable? overall reaction Back to photosystem II and structure... - orientation and why it makes sense

2 H2O -> O2 + 4 e- + 4 H+ P680 uses FOUR electrons which means 4 photons for P680 are needed for 2 water. Water is the source of electron replenishment that end up in NADPH in plants... Bacteria use acetate, succinate, malate, and sulfide as alternative electron donors (anoxigenic) ---------------------- Orientation: H2O is oxidized on LUMEN side, this makes sense because protons generated then are on the P side (thylakoid lumen) Protons are NOT pumped, but instead generated on the Lumenal P-side. Tyrosine Z (161) is a special residue that donates an electron and a PROTON from water to P680, and in the process it becomes a free radical so it will pull an electron from the Mn4CaO5 cluster... It does this 4x creating a highly oxidized Mn/O cluster strong enough to oxidize water to regenerate/reduce itself!

Salvaging 2-phosphoglycolate glycolate pathway... - what is consumed vs produced - what enzyme causes loss of co2 - Overall what is all lost - 3 organ + rxn - overall

2-phosphoglycolate has no metabolical use... - O2 is consumed and CO2 is produced! - Loss of CO2 occurs by mitochondrial decarboxylation of glycine ATP consuming process requried to recover C2 fragments from photoRESPIRATION (using O2) -> consume 1 ATP, 2 electrons (lost through H2O2, hydrogen peroxide), and lose a CO2 THREE ORGANELLES USED: 1. Chloroplast: two phosphoglycolates dephosphorylated and sent to peroxisome 2. peroxisome: cause oxidation to produce hydrogen peroxiside 3. mitochondria: lost a CO2 and oxidize a NADH overall: (2) 2-phosphoglycolate -> serine + CO2 -- 2 two-carbon culs -> one-carbon and three-carbon

Group 3: Derived from oxaloacetate (Asp, Asn, Lys, Thr) and pyruvate (Ala, Val, Leu, ILe) 1. simple for Asp, Asn, and Ala 2. Parallel paths for ... - cofactor acetyl carr...

3 non-essential amino acids made from 1-step conversion transamination with glutamate: 1. Pyruvate + Glutamate -> Alanine + alpha-ketoglutarate (seen in alanine-glucose cycle) 2. Oxaloacetate + Glutamate -> Apsartate + alpha-ketoglutarate (as shown in arginosuccinate-aspartate shunt) 3. Aspartate + Glutamate -> Asparagine + alpha-ketoglutarate ---- in general: parallel and simple pathways ILE and Leu= parallel paths start from Pyruvate - Release 1 CO2 and 1 H2O, consume NAD(P)H Mechanism: Isoleucine: start with alpha-ketoBUTYrate otherwise same mechanism Leucine: start with a second pyruvate otherwise same mechanism ***** Pyruvate is decarboxylated using TPP for both step 1 of Ile and Val (acetyl group carrier, also in glycolysis)

Stage 3 Calving cycle: regenerate ribulose 1,5-bisphosphate process... 1. aldo combine 2. FBpase 3. transket 4. final exergonic step = commit step for what? KEY NOTE on aldolase energy cofactors transke and fruc 1,6bp and where they came up before

5 of the 6 GAP are recycled back to ribulose 1,5-bisphosphate - one produced will be used in glycolysis or converted to starch Complicated series of 3C, 4C, 5C, 6C, and 7C intermediates! -- similar to pentose phosphate pathway, generates same intermediates -------------- ribose 5-Phosphate isomerase and Ribulose 5-phosphate epimerase both form ribulose 5-phoshate -------------- 1. ALDOLASE used to combine DHAP and GAP to form fructose 1,6-bisphosphate 2. Fructose 1,6-bisphosphatase forms fructose 6-phosphate 3. transketolase and aldolase keep combining intermediates! 4. Final exergonic step is Ribulose 5-Phosphate + ATP -> Ribulose 1,5-biphosphate + ADP - ****the committing step where rib 1,5-biphos will be used again in calvin cycle *****Aldolase = EXERGONIC in calvin cycle (forming DHAP and GAP is dG = 23.8, so reverse to form fruc 1,6-bp is very favorable - Transketolase cofactors: TPP -- PPP non-oxidative - fructose 1,6-bisphosphatase cofactors: Mg2+ -- glycolysis/gluconeogen

Photosynthesis... from light and CO2 to GAP-> - how many photons per GAP - alkaline made where and causes what response? - this being present in high concentration activates... -------- Diagram on pH... H+ and Mg2+ movement assist in .... lumen more ____ means more CO2 fixation

8 photons per TWO NADPH, so 6 NADPH here means 8 x 3 = 24 photons per glyceraldehyde 3-phosphate The protons move from the stroma to the thylakoid membrane (P side), so the stroma is ALKALINE conditions which triggers an accompanied Mg2+ transport from thylakoid lumen to STROMA (avoid positive charge/protons)... --> This leads to higher activity of enzymes for photosynthesis and CO2 assimilation = more ATP and NADPH which are used to make GAP ------------ H+ and Mg2+ movement assist in the activation of photophosphorylation and carbon fixation!!! -> as previously mentioned several calvin cycle enzymes are activated by increasing pH (lower protons in stroma) -> Lumen more acidic, stroma more alkaline, more CO2 assimilation GRAPH: Increase pH, higher FBPase-1 and Rubisco activity in stage 2 calvin cycle!

Chlorophyll - color - structure and prosthetic group

A type of chromophore (light absorbing molecule)-- primary pigments - GREEN pigments in the thylakoid membranes - Polycyclic, planar rings resembling heme + phytol side chains ... contain a Mg2+ in center in coordination bond What allows it to absorb visible light? - Conjugated double bond system

Other entry points pass e- to ubiquinone... (NOT using complexes 1/2) a. beta ox b. glyc-3p c. pyrimidine

A. Beta oxidation: Fatty acyl-Coa + FAD -> Enoyl CoA + FADH2 will bypass complexes I/II, give electrons to the ETF (electron transport flavoprotein). Then ETF:Q oxidoreducatase will pass electrons to form QH2 - Matrix side B: Glycerol 3-phosphate: coming from beta oxidation the glycerol molecule or reduction from glycolysis of DHAP, uses glycerol 3-phosphate dehydrogenase to form FADH2 on P side to reduce Q -> QH2 - IMS C: Pyrimidine synthesis (ch 22): dihydroorotate formed and passed electron to chain through dihydroorotate dehydrogenase with a FMN prosthetic group - IMS ALL bypass CI/II and create QH2 for complex 3 -> generate 1.5 ATP (6 H+)

Orientation of ATP synthase in different organelles *******compare/contrast

ATP synthase on outer surface (face stroma) of thylakoid membrane, PSII and PSI are facing the inside (lumen). ETC and ATP synthase all on Inner surface mitochondrial membrane. ATP Synthase faces the inside (matrix) Mitochondria: Pump protons from the inner most side (matrix, n side) to the Inner membrane space (p side, outside)... ATP generated inner most in matrix Chloroplast/photosynthesis: Pump protons from outside to inner most side (lumen, P-side)... generate ATP in outer surface (Stroma)

Lecture 22 Chapter 18-- Amino Acid oxidation and Urea-- the last catabolic reaction sequence!!!!!! Purpose of amino acids for fuel... carniv, microorg, herb, plant Three ways to obtain amino acids... die, turnov, endogen prot for energy? Are amino acids stored inside the cell?

Accumulating nitrogen compounds like ammonium are toxic, we have to get rid of them! - Strictly carnivores get 90% of energy from amino acids through meat based diet - Microorganisms scavenge aminos from environment fo fuel - Herbivores rely on carbs, a very small percentage of energy needs are met by amino acids - Plants do not use amino acids as a fuel source but degrade them to use as other metabolites Three methods to obtain and utilize amino acids for energy: 1. Diet high in protein 2. Normal turnover, synthesis and degradation of cellular proteins is constantly happening for health of metabolites 3. Endogenous proteins used as fuel during starvation or uncontrolled diabetes. (this is a bad thing) - consume enzymes, cytoskeleton, membrane proteins Amino Acids are not stored to a significant degree! Must be obtained from 1-3 above.

Before Fatty acid synthesis... Now: REGULATION What makes sense to target (early and necessary to take place) 1. palm 2. citr 3.epi/glu - slide 33 shows active vs inact (polyme)

Acetyl-CoA Carboxylase: Limit this key enzyme! Makes sense since prevents production of malonyl-CoA --> Also is the RATE LIMITING STEP in fatty acid biosynthesis Feedback/regulation of Acetyl-CoA Carboxylase 1. Palmitoyl-CoA is a negative feedback inhibitor -> Blocks Malonyl-CoA synthesis... makes sense if we have too much FA being produced we should stop it 2. Citrate is an allosteric activator -> Citrate build up means lots of energy in Krebs, lets store some! -> Also INHIBITS glycolysis PFK-1, no more making energy with too much present (futile burning), lets do FA synthesis instead! 3. Glucagon, epinephrine response (PKA) phosphorylate and INACTIVATE Acetyl-CoA Carboxylase -> Since these enzymes are short term energy needs (flight or fight, or low blood glucose response), use glucose for energy, stop FA synthesis! -> Slide 33! Phosphorylation INACTIVATES, the enzyme will actually depolymerize and leave the chain and enter solution... If these signals aren't present, carboxylase will be dephosphorylated and attached in ACTIVE chain filament, linearly Citrate -> Acetyl-CoA carboxylase Citrate -| PFK-1 glycolysis

Before, urea cycle... now linking urea cycle and TCA (krebs) cycle together... 1. Fumarate (from urea)/malate (enzyme), location produced and then shipped to 2. malate to oxal 3. oxal for energy or back to amino acid ______ to be used as donor of... uses the machinery from what pathway we previously learned (just takes a detour)? Can each cycle operate indep? 2 major fates/uses 1. need energy 2. need detox!

Aspartate-arginosuccinate shunt! 1. Fumarate produced in urea cycle IN cytosol converted to malate using cytosolic fumarase! Malate transported into mitochondria and joins pool of intermediates for citric acid cycle! 2. Malate from step one enter citric acid cycle and converted to oxaloacetate (malate dehydrogenase) 3. Oxaloactetate can continue in CAC OR it can be converted back to aspartate via transamination and to return back to urea cycle as another amino group donor! ----------- Uses machinery from malate-aspartate shuttle we learned about involved in delivering NADH equivalents for oxidative phosphorylation and ETC in mitochondrial membrane! ---------- Urea Cycle and TCA cycle operate independently! Changes depending on metabolical needs... 1. Need energy: Urea cycle fumarate can be used in citric acid cycle 2. Get rid of toxic ammonia: Resend the fumarate -> malate -> oxaloacetate -> ASPARTATE back into the urea cycle!

Mechanism to avoid having to waste energy on glycolate cycle: strategy 1: C4 vs C3 (spatial separation) Strategy 2: Temporal separation CAM ?????? Why CO2 high at night??, rewatch lecture recording

At night, air is cooler and more moist the stomata open and CO2 is absorbed and fixed to make oxaloacetate via PEP carboxykianse - oxaloacetate is reduced to malate and stored in vacuoles - during the day time malate releases CO2 via NADP-linked malic enzyme and CO2 fixation proceeds and during day STOMATA CLOSED so low O2! PEP carboxykinase also in gluconeogenesis Store CO2 at night in vacuoles, use it during the day when photophosphorylation takes place CAM = crussulacea acid metabolism -> HIGH heat, dry conditions High temp increased the solubility of O2 relative to CO2, and also decreases the affinity of rubisco for CO2. Both lead to more of rubisco's oxygenase activity, which wastes energy by producing 2-phosphoglycolate. CAM plants avoid this by keeping their stomata closed during the day when it is hot (preventing O2 from being taken in). That could also cause the problem of not having the CO2 needed for the Calvin cycle, which is why CAM plants keep their stomata open at night to take in CO2 (and store it in the form of oxaloacetate); then when they close their stomata during the day when photosynthesis begins, CO2 can be taken from oxaloacetate for the Calvin cycle without needing to open stomata for gas exchange.

The Ketogenic amino acids from previous notecard can be degraded to... gen idea (shared path) succ coa keton where else they appear

Blue: Gluconeogenic Red: Ketogenic! General idea: Degradation of an amino acid will result in overlapping pathways where part of the skeleton is gluconeogenic and part is ketogenic and goes to their corresponding places. Propionyl-CoA -> Succinyl-CoA seen here! Uses the same enzymes as fatty acid synthesis of odd number chain Ketone body production: Low oxaloacetate (lots of gluconeogenesis and low glycolysis, from low carb diet)

Mechanism to avoid having to waste energy on glycolate cycle: strategy 1: C4 vs C3 (spatial separation) - mechanism 1. mesophyll (pep oxalo) 2. malat 3. oxid to pyr and form 4. stage 1 - note on temp and energy req - which is more likely to do better in heat - shared enzymes Strategy 2: Temporal separation

C3 plants: Normal, first step is ribulose to 3-phosph C4 method: Have an adiditional step before rubisco! Plants bypass fixation step by fixing CO2 into a FOUR carbon compound... spatially separate CO2 fixation from rubisco activity! Hide rubisco in bundle-sheath cell adjacent to mesophyll leaf cell where [CO2]>>[O2] Special enzyme: pyruvate phosphate dikinase-- uses two ATP equivalents C4 mechanism: 1. CO2 is captured in mesophyll cells using PEP carboxylase to go from PEP to Oxaloacetate (4 carbons) 2. Oxaloacetate reduced to -> malate with malate dehydrogenase to enter the bundle-sheath cell 3. In bundle sheath cell, malate oxidized to pyruvate which releases a CO2 and makes NADPH! 4. Bundle-sheath not exposed to cell so its very highly saturated with CO2 and way less likely to undergo oxygenation/wasted energy, rubisco will catalyze stage one TEMP: Increase temperature helps overcome the large energy barrier required for this reaction to take place. -> C4 plant will likely do better in HEAT than C3 plants -> often weeds! like crabgrass Enzymes reappearing: Malate dehydrogenase-- from gluconeogenesis, malate-aspartate shuttle Malic enzyme-- reforming oxaloacetate (anaplerotic rxns) Pep carboxylase: Anaplerotic rxns

Most org FA synth/oxid it occurs... - therefore, must - TRUE cost plants it occurs... therefore, transp.

CELLULAR LOCATION: Most Organisms: - Fatty acid oxidation: Occurs in the mitochondria. Acetyl-CoA accumulates here! - Fatty acid synthesis: Occurs in the cytosol! Makes sense to separate -> So does nucleotide, glucose, and amino acid synthesis! Therefore, Acetyl-CoA largely in mitochondria and must be transported to the cytosol! This requires 2 ATP, so it ends up coming out to 3 ATP required per 2 carbon unit added! -> Inner mit mem impermeable to Acetyl-CoA so must use transport (next slide) Plants: - Occurs in the stroma of chloroplasts -> makes sense since key reactant NADPH is generated in stroma via non-cyclic light reactions

Nitrogen Fixation: reaction easy, dG, why so high Ea complex overall reaction - number ATP? Electrons? Seen before steps and important parts... 1. pyruv -> ac - where seen 2. fd -> dini red - where seen 3. dinit red -> dinit -> red N2

Carried out by the nitrogenase complex! N2 + 3 H2 -> 2 NH3 - dG= -33.5 KJ/mol (very favorable) - Super high activation energy due to breaking of triple bonds = super slow reaction and needs ATP N2 + 10 H+ + 8 e- + 16 ATP -> 2 NH4+ + H2 + 16 ADP + 16 Pi STEPS: 1. Pyruvate + CoA oxidized to form acetyl-CoA -> Saw this in stage 1 aerobic respiration pyruv -> ac-CoA - not always pyruvate, some reaction reduces ferredoxin though 2. The electrons are captured not by NADH, but by FERREDOXIN -> seen before in photosynthesis PSI 3. These reduce dinitrogenase reductase to reduce dinitrogenase complex to then reduce N2 -> 2 NH4+

The ATP Synthase Paradox ----- Fo is a proton-couple rotor (motor)... binding sites contain...

Catalytic formation of ATP is reversible: - dG'˚ for the reaction approaches 0 KJ/mol with help of the F1 enzyme - dG'˚ for rxn on its own is + 30.5 KJ/mol how can enzyme make this an unfavorable REVERSIBLE reaction? Change affinity: The enzyme has extremely high affinity for ATP (10^-12 M) and very low ADP is only (10^-5 M) --- this difference in Kd results in about 40 KJ/mol whereas ATP synthesis only requires 30-50 KJ/mol --------- Fo is a rotor with binding sites that feature Aspartate (or glutamate... negatively charged) which bind PROTONS - this turns the HANDLE - c domains are the rotor, a domain is the proton transporter

End e- transport creating H+ gradient.... NOW: H+ gradient used to make ATP ------------ Chemiosmosis definition e- transport set up .. this drives ... couple X to Y ------------- Chemiosmotic theory: H+ motive force drives ATP production

Chemiosmosis is the movement of ions across a semipermeable membrane bound structure, down their electrochemical gradient. Electron transport (chain) set up proton motive force, proton motive force drives synthesis of ATP through coupling H+ flux to phosphorylation -------------------- ADP + Pi -> ATP highly thermodynamically unfavorable! -> phosphorylation is a result of proton motive force... NOT direct reaction between ADP and some high energy phosphate carrier!

Photosystems are... photochem rxn centers chemical pigments vs antenna those able to convert to chem en process? light -> chemical by sep char img shows? (anten/rxn cent) What happens if chlorophyll is in solution? (primary vs acces pigments)

Chlorophyll and other pigments are arranged in functional arrays within photosystems! All pigments absorb light, but only a few CHLOROPHYLLs can transduce light energy into chemical energy, but the ones that do are associated with Photochemical reaction centers (chlorophyll a)! versus Pigments just absorbing light and transmit it to neighboring cells are "antenna" molecules part of the light harvesting complex and surrounding the reaction center. Process of light->chemical - Photochemical reaction center converts energy of a photon into a separation of charge which initiates electron flow. The top image shows a reaction center in a photosystem, and the light harvesting complex is the green rectangle in the membrane which is a protein bound to chlorophyll where light is absorbed. If chlorophyll is in solution/free floating, the energy is lost as heat or fluorescence since it is not associated/cannot pass energy to neighboring antenna to filter to reaction center Primary pigment: make up the reaction center (special pair) -- chlorophyll a Accessory pigment: Complementary and give the range of colors -- carotenoids, lutein, phycobillins etc.

Pigments and light absorption - what are chromoph? abosrbing en (how much required?) Releasing en (key note on efficiency... emit ...

Chromophores: A type of pigment, which is a light absorbing molecule with different energy states - often conjugated double bonds 1. Absorbing light energy: - Absorbing a photon of light will raise the chromophore to the next level - Photon absorption requires exactly enough energy to raise energy states, electrons literally jumps levels 2. Releasing energy: - Chromophore can convert back to ground state when energy releases as heat, light, or work - emitted light/fluorescence will ALWAYS be less in energy than what was absorbed (never completely efficient process) - Excitons may be transferred to nearby molecules/passed off!

Cytochrome b6f in depth look at its function in PSII/PSI translocation three components net pump Slide 34 ch20P1, image of protein make up/chromophore (color) make up

Chytochrome b6f links PSII and PSI and translocates protons into the thylakoid lumen. - undergoes a Q cycle similar to ETC... - 2 PQH2 + Q -> 2 Q + PQH2 (net consume a PQH2) - electrons one at a time travel up to reduce plastocyanin and the other down to reduce PQ (repeat one more time). NEXT: electrons are carried from plastocyanin to PSI for next cycle three key components: - Cytochrome b6 (takes electrons from plastoquinone) - RIESKE Iron-sulfur protein (helps transfer electrons, histidine and cysteine residues) - Cytochrome f (passes electrons to plastocyanin) NET: 4 H+ pumped to P side (thylakoid lumen) per pair of electrons!

Different wavelengths of light graph: - complementary - energy/wavelength - changing seasons - not all same color tree - note on clorophyll

Complementary pigments cover the whole spectrum so all colors in visible light spectrum are seen. Key: Lower wavelength = more energy! Starting left, colors will drop energy and flow right, but cannot flow upwards in energy/left! Changing seasons and leaves: As season changes, less light and cold weather causes leaves to breakdown pigments to store energy, so secondary pigments of lower energy being to appear as different colors. Not all trees are the same green: Mixture of pigments based on environment results in exact coloration Note: Chlorophyll a and b absorb different wavelengths and are not even in the green region.

Regulating Oxidative phosphorylation... acc con reg of oxid phos by mass act Diagram with regulations...

Complete oxidation of glucose produces 30-32 ATP - aerobix oxidation and transfer of e- to O2 is producing vast majority of ATP (comparatively: Anaerobic glycolysis produce 2 ATP ... fatty acid palmitoyl-CoA produce 108 ATP) How things are regulated: - Cellular respiration (O2 oxidation) is limited by [ADP] ---- ACCEPTOR CONTROL: How able ADP is to accept and turn into ATP... based on ratio Mass action ratio: [ATP]/[ADP] (or AMP) .. P/reactant - at rest the ratio is high and maximally phosphorylated - Under stress, ATP levels drop and so do ratios - The ratio provides a sensitive filter for [ATP] ATP/ADP ratio regulates: oxidative phosphorylation, CAC, glycolysis, pyruvate oxidation Most control is feedback inhibition! - as ATP hydrolysis increases so does electron transfer, pyruvate oxidation (generate acetyl-coa), and ATP synthesis All increase rapidly and proportionately!

1. NADH DH 2. Succinate dehydrogenase- complex II 3. Ubiquninon:cytochrome c oxidoreductase-- Q Cycle - ubiq used red C - contain fe/s, c bs, cs - 4 total - switch 2 e to 1 FUNCTION of Q cycle 1. move H - re-red and oxid 2. pass to IV

Complex III undergoes the Q cycle->> 4 H+ transported to the inner mitochondrial membrane! - 2 electrons from ubiquinol QH2 are used to reduce 2 molecules of Cytocrhome C (single e- carrier) - contains iron-sulfur clusters, cytochrome bs, and cytochrom cs - 4 total hemes - functions as a switch from 2 electron carriers (NADH, FADH2, Q) to 1-electron only carriers (cytocromes, Cu) Q cycle FUNCTIONS: 1. Move 4 H+ to the intermembrane space (2 come from QH2 and 2 come from the matrix) - 2 QH2 oxidized, release protons into IMS, one QH2 gets re-reduced, net transfer four protons per reduced coenzyme Q 2. Pass 2 electrons to Complex IV

Group 2: cysteine universal sulfide donor 0. start w 1. APS 2. PAPS 3. Pap 4. sulf to be used s2 below 0. serine 1. 0-ac 2. add w s for cys

Cysteine = form NADH, use up 4 NADPH and 2 ATP and 1 Acetyl-CoA - Two amino acids have sulfur: Cys and Met - PAPS = universal sulfide donor!!!!!! - 2 ATP, 3 ATP equivalents! Special mechanisms for sulfur... 1. Production of sulfide - use sulfates (SO4 2-) in environment, activated by ATP - So4 2- reduced using 8 total e- to S 2- 2. Serine activated by acetyl CoA, acetate replaced with sulfide 3. Uses 1 acetyl-coa, 2 ATP, 4 NADPH MECHANISM: start by forming sulfide.... 1. ATP + SO4 2- -> Adenosine 5-phosphosulfate (APS) ATP-sulfurylase - adenylylation 2. APS + ATP -> 3'-phosphoadenosine-5'-phosphoadenosine (PAPS) - 2 phosphates one one cul!! 3. PAPS + NADPH -> PAP + NADP+ PAPS reductase 4. PAP + 3 NAPDH -> S 2-(sulfide) + 3 NADP+ Sulfide Reductase - see sulfide added to O-Acetylserine below in step 2! -------- 0. form serine 1. Serine -> O-Acetylserine serine acetyl-transferase 2. O-Acetylserine + S 2- -> Cysteine + CH3OO- O-Acetylserine (thiol) lyase

SLIDES 9-12 ch 19 Universal electron carriers: Key Qs 1. transfer one or two e-? 2. Prosthetic group? 3. anabolic rxn? 4. other A. nictoniamide 4. Cross mit membrane? B. Flavin C. ETF D. ubiq/coq 4. oxidized name to reduced name 5. soluble... conj dicar, 6. Move freely, present in E. cytoch 4. heme make up 5. inner mi me 6. spectra shows F. Fe-S clu 4. Fe is assoc w cys s 5. Rieske

Dehydrogenases capture e- and transfer them to... A. Nicotinamide nucleotides: NAD+ and NADP+ 1. transfer TWO e- only (hydride ion and poton) 2. Cofactors/Coenzymes 3. NADPH anabolism, NAD+ catabolism 4. Do NOT cross the inner mitochondrial membrane B. Flavin Nucleotides: FAD and FMN 1. Transfer one e- or two e- (can do one at a time), can enter semiquinone state 2. Prosthetic group- permanently associated w/ protein 3. FAD = Catabolism C. Electron transferring flavoprotein (ETF) (collects electrons from beta-oxidation of fatty acids using flavin nucleotides) D. Ubiquinone (Coenzyme Q): membrane bound universal e- carrier 1. Transfer one e- (result in radical intermediate) OR two e- 2. Coenzyme 3. Catabolism 4. Ubiquinone -> Semiquinone radical -> ubiquinol (reduced with 2 e-/2 H+) = QH2 5. Lipid-soluble (long non-polar tail), conjugated dicarbonyl, like a lipid 6. CoQ is present and uses lateral diffusion with oxidative phosphorylation chain complexes I and II to deliver electrons to III E. Cytochromes 1. ONE only electron carrier 2. Heme group is a prosthetic group (tight but non-covalent)! 3. Catabolism 4. Heme groups contain Fe (iron) 2+ or 3+ in the porphoryin ring 5. most cytochromes are integral protein components of inner mitochondrial membrane 6. Color on spectra shows if oxidized or reduced cytochrome is present! F. Iron-Sulfur Clusters: 1. ONE electron carriers 2. Prosthetic groups 3. Catabolic 4. Iron is associated with inorganic sulfur or Cysteine amino acid sulfurs 5. Rieske iron-sulfur cluster: Fe associated with 2 His instead of 2 cys

Proton-Motive force remember eq for calc dG based on electrochemical gradient! (Z, dpsi) electrochemical potential calculation:

Electron transport chain creates electrochemical proton gradient through... 1. ACTIVE transport protons across inner membrane. 2. Chemcially remove protons from matrix by reduction of Q to QH2 (lower H [ ] in matrix) ADP -> ATP req dG = +50 KJ in cell conditions electrochemical potential calculation: dG = RT ln [P]/[S] + zF∆ψ - z = charge of solute (+1 if Na) - ∆ψ = Membrane potential in VOLTS (usually -50 mV.. -.05 V) - F = faradays constant = 96,500 J/V*mol

The Cellular Metabolic Hub: Citric acid cycle and entry points

Entry into the citric acid cycle with MORE than two carbons yeild a net increase in the amount of citric acid cycle intermediates! These can then be used for synthesis of other metabolites... AEROBIC metabolism Either use amino acid carbon skeletons to produce glucose or send it into the citric acid cycle to generate energy or other metabolites!

FA sytnehsis requires fatty acid synthase enzyme 7 subunits FAS-I - track c15/c16 vs FAS-II --- what organisms, leads to?

Fatty Acid Synthase: SEVEN catalytic subunits/different active sites... C-shape 7 poplypeptides, 3 accessory proteins: 1. KS (Ketoacyl Synthase) 2. ACP (Acyl Carrier protein) 3. KR (ketoacyl reducatse) 4. DH (dehydratase) 5. ER (Enoyl-ACP reductase) 6. MAT (malonyl-acetyl-CoA transferase) 7. TE (thioesterase-- to release final product) KS and ACP have a thiol group that carry the acetyl/malonyl groups the whole way through! FAS-I: Single polypeptide chain in vertebrates... - leads to a single product (palmitate 16:0) - C-15 and C-16 are derived from the one and only Acetyl-CoA used to prime the reaction! FAS-II: Made of separate, diffusible enzymes - Leas to many different characteristic fatty acids - Can make sat/unsat/branched/varied lengths etc. - Mostly in plants and bacteria!

Before regulation, now elongate, desaturate, phospholipid synt--- Fatty Acid Elongation System - adds - simila mech - consumes X nadph and Y atp Branch points, arachid/essential FA's

Fatty acid elongation system: - Add acetyl groups from malonyl-CoA - Similar mechanism... condensation, reduction, dehydration, reduction - consume another 2 NADPH (and ATP to form malonyl technically 3 ATP to transport) Occurs: In the mitochondria and Smooth Endoplasmic Reticulum Branch points: Elongate/Desaturate - mammals cannot make as many different modifications! - We rely on PLANTS to make Omega 3/6 fatty acids which we can then convert/process to make key intermediates like Arachidonate which is a precursor for eicosanoids for signaling and anti-inflammatory!

Regulating Purine synthesis! 1. AMP 2. GMP 3. IMP

Feedback inhibition: Monophosphate inhibit production of more monophosphates!!!! 1. AMP will inhibit making itself 2. GMP will inhibit making itself and another uphill (sequential inhibition) 3. If GMP and AMP block IMP catabolism it will build up and be the DOMINANT regulator of PRPP-> 5-phosphoribosylamine

We focus on three groups of amino acid synthesis... Group 1: Alpha-ketoglutarate (CAC) Group 2: 3-phosphoglycerate (glycolysis) Group 3: Pyruvate/oxaloacetate (Glycolysis/CAC) ----------------------------------- Group 1 focus in... (P/R) what overall is produced? Costly? path it comes from! energy requirement overview mechanism these two follow!

G1: Alpha-ketoglutarate: Glutamate, glutamine, proline, arginine G2: 3-phosphoglycerate -> Serine -> Glycine and cysteine G3: Pyruvate -> VAIL (valine, alanine, isoleucine, leucine) Oxaloacetate -> MALT (methionine, Asparagine, Lysine, Threonine) Group 1: Citrate ->-> Alpha-ketoglutarate -> Glutamate-> Glutamine, proline, arginine What is produced?!?! - Lots of ADP, NADP+, and some AMP... some alpha-ketoglutarate... and some fumarate from urea cycle Arg ---- very costly, some of products used in other paths -> Previously covered glutamate/glutamine coming from alpha-ketoglutarate! Now Proline/arginine! - Requires 2 NADPH/NADH and 1 ATP - arginine can be made into proline in mammals General mechanism occurring throughout: 1. Costly in energy (anabolism usually is) 2. Phosphorylation and then reduction 3. Simple transformations 4. Amino acids synthesized with overlapping pathways

Group 2: Derived from 3-phos ... ser, gly, cys --- omit cysteine until next slide - dephos - oxid - simple conv 1. 3-pgt . 3phhydpyr 2. add in glu 3. dephis 4. (for gly, uses familiar C carrier)

General idea: simple conversions between amino acids serine and Glycine (one step from serine -> glycine) - OXIDIZE 3-phosphoglycerate to 3-phoshohydroxypyruvate - dephosphotylation to produce serine 1. 3-phosphoglyerate + NAD+ -> 3-phoshohydroxypyruvate + NADH + H+ 3-phosphoglycerate dehydrogenase 2. 3-phoshohydroxypyruvate + Glutamate -> alpha-ketoglutarate + 3-phosphoserine phosphoserine aminotransferase 3. H2O + 3-phosphoserine -> Pi + Serine Phosphoserine phosphatase .......... 4. Serine + H4 Folate -> THF + H2O + Glycine serine hydroxymethyl-transferase - uses THF

Regulation of Glutamine Synthetase ... incorporating nitrogen into glutamine from glutamate! -- central point types 1. cov 2. all

Glutamine is the central entry point for reduced nitrogen into most metabolic pathways. --- this enzyme = trying to include more nitrogen into our systems, so we would want inhibitors to be molecules signaling we are breaking down nitrogen (like carbamoyl phosphate) Two types of regulation: 1. Covalent: phosphorylation (Adenylylation) will cause the enzyme to become even more sensitive to the inhibitors! 2. Allosteric: Additive! - carbamoyl phosphate - Tryptophan - AMP - CTP - Histidine - Glucosamine 6-phosphate -> Each turns down the enzyme activity a little bit, add up each constituent to produce the whole!

NADH Shuttle number 2: Glyc 3-P shut ... in where? Number ATP produced with this step?

Glycolysis and other mechanisms produce NADH in CYTOSOL... need to transport across Glycerol 3-Phosphate Shuttle (in the skeletal muscle and brain) - some comes from beta-oxidation of fatty acids Cytosol G3PDH: Glycerol 3-phosphate dehydrogenase moves electrons from NADH to glycerol 3-phosphate Mitochondrial G3PDH: Moves electrons from glycerol 3-phosphate into respiratory chain -- NOTE: 2 of the SAME enzyme used! ... Upon oxidation of glyercol 3-phosphate, it turns to DHAP (can be used in glycolysis) Produces 30 ATP from a glucose

Transporting ADP and Pi into matrix where the F1 subunit is in need of it.... Two types of transport: 1. antiport... fueled by (+) 2. symport... fuel (chem) and accounting for total H+ used with ATP generation... P/O ratio...

Goal: Move ADP into matrix, move ATP out of the matrix 1. Adenine nucleotide translocase antiporter - exchange ADP3- for ATP4- in the intermembrane space - ATP exits matrix, ADP enters FUELED BY: - the charge gradient! ATP 4- exits into the P-side (+ charge) which is favorable. Follows its electrical gradient 2. Phosphate translocase symporter - H+ and H2PO4- (Pi) enter into the matrix Fueled by: - H+ chemical gradient brings Pi against its gradient ----- NOTE: this decreases the established electrochemical gradient by one more proton! Accounting for protons: P/O ratio = number of ATP made per oxygen atom (2 electrons) consumer 1. Number of protons pumped out in ETC A. NADH was 10 protons B. FADH2 was 6 protons 2. Number of protons needed to drive synthesis of ONE SINGULAR ATP A. if 9 c-subunits, 9 protons per cycle, total 9 protons / 3 atp = 3 protons per ATP B. plus one proton to get a Pi inside! 3 + 1 = 4 Protons Proton based P/O ratio: NADH: 10/4 = 2.5 FADH2: 6/4 = 1.5 .... 12 c-subunits: - 10 NADH, 6 FADH2 - 12 H+/ 3 ATP = 4 ... plus one transporting Pi = 5 protons 10/5 =2 6/5 = 1.2

Group 1 vs 2 vs 3

Group 1: Requires 2 NADPH/NADH and 1 ATP - phosphorylation then reduction - similar pathways but arginine must be blocked first - Arginine uses urea cycle ornithine path-> Arg Group 2: glycine/serine Produces NADH - One step conversion between serine and Glycine - OXIDIZE 3-phosphoglycerate to 3-phoshohydroxypyruvate - dephosphotylation to produce serine cysteine: uses 2 ATP, 4 NADPH and 1 acetyl CoA Group 3: Asp/Ala/Asn = simple aminotransferases with Glu ILE and Val= parallel paths start from Pyruvate - Release 1 CO2 and 1 H2O, consume NAD(P)H - use TPP cofactor acetyl carrier

Before: Incorpoating amino's into amino acids and making useful forms of N2! NOW: synthesizing amino acids Overview - source of N is... - three processes and interm overview in chart - bacteria have ____, humans.... - paths inter, also are sevrl

In amino acid synthesis, the source is again Glutamine/glutamate! - add N to intermediates from glycolysis, PPP, citric acid cycle! - Bacteria synthesize all 20, humans require some in diet (essential amino acids) - Amino acids have interconnected pathways, and some have several synthesis pathways Glycolysis: - Pyruvate: Alanine, valine, leucine, isoleucine - 3-phosphoglycerate: Serine, glycine, cysteine - PEP (shared with PPP!!): Tryptophan, phenylalanine, tyrosine PPP: - Erythrose 4-phosphate (shared with glycolysis!!!): Tryptophan, phenylalanine, tyrosine - Ribose 5-phosphate: Histidine CAC: - Alpha-ketoglutarate: Glutamate, glutamine, proline, arginine - Oxaloacetate: Aspartate, asparagine, methionine, theronine, lysine green = non-essential

Phycobillins strutucre, diff from chloro similar roll tide ----- Carotenoids (slide 13 ch 20) 2 examples beta/lut - present where?

In chloroplasts of red algae and cyanobacteria Structural difference from chlorophyll: - NON-cyclic ring structure, no heme nor central Mg nor metal compound. - extended polyene chains are similar to chlorophylls Red tide (alabama) named after the red algae which take over beaches showing the water is toxic ------------------ Carotenoids: yellow, red, or purple pigment... present in thylakoid membranes (its a LIPID!!!!) - Beta-Carotene: red/yellow = orange - Lutein (xanthophyll): Yellow Carotenoids are COMPLIMENTARY to chlorphyll (help extend the range of light it can absorb/respond to)

Sucrose synthesis from the GAP made in chloroplasts calvin cycle... - inn chlo memb imperm to - solution = 2 types of antiporter exprot only (atp) vs scur import(run in cycle) = equiv

Inner chloroplast is IMPERMEABLE to phosphorylated compounds! Pi-triose phosphate antiporter: EXPORT DHAP direction ONLY: will exchange a DHAP out of the cell, for glycolysis/gluconeogenesis to convert to sucrose, and a Pi into the cell forATP synthesis! - stroma = NADPH Full cycle: Export equivalents of ATP and NAD(P)H into the cytosol to be used for energy - Cytosol=NADH

NOW: three types of rxn centers... 1. Bacterial Type II 2. Bacterial Type I -- aka - cyclic vs noncyclic? -Fd:nadhreduc - found in? 3. Plant Reaction Centers (type II-type 1 in series)

Iron-sulfur reaction centers (Type I): not always cyclic! - bacteria found in hot pools, green sulfur bacteria Cyclic: 1. P840 reaction center excites 2. Electrons passed to quinone 3. Quinone uses cytochrom bc1 complex and Q cycle for generating a proton gradient and then passes electrons to create cyclic reaction center reactions non-cyclic: 1. Excited P840 decreases in E˚' 2. electrons passed to FERREDOXIN 3. Electrons then passed to feredoxin:NADH reductase 4. electrons used to generate NADH electron carrier How are electrons replaced? 5. HS2 is oxidized to H2SO4 to give electrons to reaction centers

Research sidelight: Protein NMR uses and signals!

Isotope 15N, need an odd number to visualize using NMR... - feed bacteria 15NH4 and they incorporate this into all the amino acids! - NMR spectra can be taken, each point is a peak/amino acid! Change the 15N you feed the bacteria (Serine or Glutamate, or proline, etc.!!!) and you can really pinpoint which amino acids each spectra is within the protein Feed glutamate: All amino acids basically show since its used to make almost all Feed serine: Must either be serine, glycine, or cysteine... can help narrow the peaks down to the individual amino acid!

Light activates four enzymes (relies on disulfide bonds) - residue? 1. fd red 2. thiro 3. target enz

Light activates four enzymes via electron-driven reduction of cys-cys crosslinks - photophosphorylation results in a REDUCED environment as all reaction centers charge and donate electrons... this results in disulfide bonds between cysteine residues breaking open and becoming active!!! 1. Feredoxin will be reduced when in presence of light 2. it can pass electrons to thioredoxin enzyme which will be reduced and activated 3. Thioredoxin can then reduce target enzyme to activate calvin cycle and consume ATP and NADPH - target enzymes: see image (all from calvin cycle stage 2 or 3)

More on Rubisco... Mg2+ significance and structure Cryo-EM 5 bonds steps to carbomylation key on carb lys and how its regulated!?

Magnesium is positively charged and held in Rubsico by negative charged side chains like aspartate, glutamate, and a carbamoylated lysine! - requires the carbamoylated lysine to be active Mg2+ brings together the reactants in the proper orientation and stabilizes the negative charge that forms upon the nucleophilic attack of enediolate to CO2. 5 salt bridges... 1. carbamoylated lysine 2. aspartate 3. glutamate 4. CO2 (reactant) 5. Rib 1,5-BP (reactant) -------- Lysine-201: steps to cabromylation Carbamoylated lysine just means the lysine is modified to have a CO2 group attacked to the nitrogen side chain... 1. Lys-201 is inactive (inaccessible) until rubisco activase exposes the lysine to environment 2. Carbomylation is spontaneous and will form active rubisco 3. Mg2+ now will link in the active site and recruit reactants regulation: 1. Rubisco activase: changes rubsico conformation to expose lys-201! LIGHT triggers this! It requires ATP. -> makes sense because light means photophosphorylation and NADPH/ATP around allowing for calvin cycle to take place Overall... no ATP is needed for the reaction, but ATP is needed to form activated rubsico!

Formation of essential substrate for FA anabolism: Malonyl-CoA Reaction enzyme (name makes sense, think what rxn is happening to what substrate) Three subunits of enzyme/X active sites... all are on one ______ ___ - prosthet group - in general this happens - X is required

Malonyl-CoA is a key 3 carbon building block for fatty acid synthesis. Acetyl-CoA + Bicarbonate -> Malonyl CoA Acetyl-CoA Carboxylase Acetyl-CoA Carboxylase Reaction: - enzyme has three subunits - Animals have three subunits where all are ONE polypeptide chain - ATP is required for the biotin to transfer the CO2! Bicarbonate transfers CO2 to Biotin, which transfers the CO2 to Acetyl-CoA to form malonyl-CoA Three Subunits: TWO active sites 1. Biotin Carrier protein: one unit has PROSTHETIC biotin covalently linked to LYSINE, a CO2 carrier group 2. Biotin Carboxylase active site 3. Transcarboxylase active site Steps: 1. ATP hydrolysis allows for the activated bicarbonate molecules (via phosphoryl transfer) to be attached to the biotin carrier protein USING the biotin carboxylase subunit! 2. Biotin-CO2 unit will now actually "swing" over using the flexible tether lysine molecule/some of the biotin atoms branch. It will then bind the transcarboxylase subunit to transfer a CO2 to Acetyl-CoA Summary: Biotin attach CO2 from bicarbonate at biotin carboxylase -> swing to transcarboxylase -> CO2 added to Acetyl-CoA to form Malonyl-CoA

Regulation of competing pathways of Fatty acid synthesis/Oxidation... How is it controlled?

Malonyl-CoA is required for the start of Fatty acid synthesis! So it is a good point to act as a regulator! Beta-oxidation of FA: Carnitine Acyltransferase I enzyme to transport acetyl CoA into the cell INHIBIT this enzyme to prevent futile cycling! Do not put acetyl coa in mitochondria for breakdown when we have enough energy and instead should be storing!

Non-dietary amino acid metabolism: aminos coming not from stomach nor muscle, but from all other tissues! glut synthet 1. glutamyl 2. gln ->trans 3. glu ->excrete enzymes for 1/2 and 3 tracking ammonia for urea cycle!

Metabolism of amino acids by all other tissues requires the toxic ammonia to be carried off, but the carbon skeleton will be saved for CAC. How do you transport ammonia to liver to deal with? - Glutamine syntetase --- hydrolysis results in ammonia in Liver!!! KEY: Each amino acid transport requires 1 ATP, so there is a cost 1. L-Glutamate + ATP -> ADP + γ-glutamyl-phosphate 2. γ-glutamyl-phosphate + NH4+ -> Glutamine + Pi ---> Transport via blood to liver as glutamine, under low metabolic load! 3. Glutamine + H2O -> Glutamate + NH4+ ---> Ammonia excreted as urea 1+2 = Glutamine synthetase 3 = glutaminase enzyme GLUTAMINE where ammonia goes... 1. Break a free ammonia which is captured by bicarbonate to form carbamoyl phosphate 2. Now as glutamate, release nitrogen to oxaloacetate to generate aspartate to enter urea cycle!

Fatty Acid Desaturation techniques 1. Mono-unsaturated - Two common ones - mammals? - requires - Enzyme used ... X is reduced Y/Z are oxidized 2. Poly-unsaturated slide 39

Most common are palmitoleate 16:1 (delta^9) and oleate 18:1 (delta^9) -- Mammals can only introduce double bonds at delta^9!! (between C9/C10) - requires NADPH - Double bond is introduced by oxidation using mixed function oxidase (fatty acyl-CoA desaturase) Components of this reaction: 1. NADPH + H+ oxidized to NADP+ 2. Cyt B5 reductase FLAVINS FAD reduced and then oxidzed when passing 2 e- and H+ to TWO cyt b5 iron (ferrous) carriers 3. Cyt B5 reduces O2 and generate monounsaturated product Both NADPH gives up 2 e- (which are given to O2 via Cytochrome B5) and Saturated fatty acyl-CoA give up 2 e- and so are oxidized giving up 4 e- and 4 protons total --> O2 is reduced forming 2 H2O Similar to the flow of e- in the ETC! Nicotinamide -> Flavin -> Iron + cytochrome -> O2 __________________________ Poly-unsaturated: - Mammals can only make double bond at Delta 9 - Plants can make them at delta 12 and 15 - humans can take plants and modify them to linoleate (18:2 ∆9,12) and linolenate (18:3 ∆9,12,15) <-- essential fatty acids! ingestion, transformed to ex. arachnoid (18:2 ∆5,8,11,14)

_____________ Transports ammonia from skeletal muscles to the liver... Process: gluc/A cycle - SIMILAR TO WHAT CYCLE WE HAVE SEEN BEFORE?!?!

Muscle cells have no spare ATP since they are all devoted to muscle contraction, this metabolic burden is imposed on the liver to use ATP to convert alanine back to pyruvate and glucose! Glucose-Alanine Cycle: 1. Glycolysis forms pyruvate and muscle protein breakdown forms glutamate 2. Pyruvate and glutamate can be used to form Alanine using alanine aminotransferase 3. ALANINE transported from muscle to liver through the blood stream 4. In the liver, ATP is used to convert alanine to pyruvate via alanine aminotransferase, then to glucose which can go back to muscle! Liver: Alanine -> pyruvate -> glycose (gluconeogen) - KEY: Muscles shift the burden of dealing with ammonia to the liver! Similar to the CORI cycle: Before we transferred glucose to muscle, and pyruvate to liver to reduce back to glucose! Cori is used when we have enough glycogen but no O2... Glucose-alanine used if we are out of glucose!! Muscle consumes energy and makes waste, liver deals with waste to synthesize energy.

Balance sheet electron transport and how we get ATP dE/dG... each electron pair

NADH oxidation rxn: dE'˚ = 1.14 V and dG'˚= -220 kj/mol Net energy from both reactions is mostly used to pump H+ from the matrix to IMS! Each electron pair: (NADH) 4 H+ are pumped out by complex I (FADH2 complex 2 is 2 H+) 4 H+ are pumped out by complex III 2 protons pumped out by complex IV --- NADH = 10 H+ FADH2 = 6 H+ CI -> CIV ... 1.5 atp NADH + 11 H+ + 1/2 O2 -> NAD+ + 10 H+ (P-side) + H2O CII -> CIV ... 1.5 atp FADH2 + 6 H+ (N-side) + 1/2 O2 -> FAD + 6 H+ (P-side) + H2O

First: overview on Cytochrome C The order of electron ---- 1. NADH DH 2. Succinate dehydrogenase- complex II 3. Ubiquninon:cytochrome c oxidoreductase-- Q Cycle 4. Cytochrome oxidase-- complex IV - number subunits - two heme groups - copper ions - copper complex

NADH/FADH2 -> QH2 -> Cytochrome C (2nd e- carrier) -> O2 Soluble heme containing protein in IMS, can be ferrous (Fe 3+) or Ferric (Fe 2+ reduced) Cytochrome C carries a single electron from cytochrom bc1 from complex III to cytochrome oxidase in complex IV. ---------- - Mammalian cytochrome oxidase is a membrane with 13 subunits - Contains two heme groups: a and a3 - contains two copper ions: 1. CuA: two ions that accept electrons from Cyt C ... 2. CuB: bonded to a heme a3 forming a binuclear center that transfers four electrons to oxygen Copper complex: NO IRON -- copper cluster Electrons are charged in CuA two Cu Ions as Cu1+Cu1+ but when oxidized they are Cu1.5+Cu1.5+

Ribonucleotides generate (UMP, CTP, AMP, GMP) can be converted to Dexoyri

NDP -> dNDP Ribonucleotide reductase --> which is activated by thioredoxin Requires a pair of H+ originally donate by NADPH - pathway involves FADH2 and THIORDEXIN (as seen in photosynthesis, Fd activated thioredoxin which could activate enzymes in the calvin cycle (stages 1-3) ) - or path involves glutaredoxin and GSSG-> 2 GSH 1. NADPH -> FADH2 -> Thioredexin -> Ribonucleotide reductase -> dNDP

Bacterial type II Pheophytin-Quinone reaction center mechanism steps 1. rxn center excited 2. pass to ph 3. pass to Q 4. Q cycle release 5. recycle structure... slide 26 ch 20P1 some similarities to CIII... Compare/contrast

Note on the graph: Top is low reduction potential, bottom is higher so as they excite they move up to and become more negative becoming less electron affinity, so they can now flow to higher/more positive reduction potentials. (Jump from .5 -> -.75) PURPLE BACTERIA CYCLIC PATHWAY 1. P870 is excited, becomes more negative. 2. Electrons passed to reduce pheophytin 3. Pheophytin passes electrons to reduce quinone to a semiquinone intermediate just like complex III mitochondria, and then reduce coenzyme Q to Q(B)H2 (similar to QH2 of CIII). 4. QbH2 will then be used by the cytochrome bc1 complex. This complex undergoes a Q CYCLE (2 QbH2 + Q -> QbH2 + 2 Q so net 1 used up) and releases the 4 consumed protons on the other side of the membrane (P side) while also reducing 2 cytochrom c2 molecules. - protons create gradient here to generate ATP! 5. Cytochrome c2 will release electrons to flow back to the reaction center p870 to be able to undergo another round!!!! STRUCTURE: The structure shows close quarters between the different electron carrier intermediates allowing for rapid exchange of the electron in channel fashion.. P870 -> Pheophytine -> quinone -> Cytochrome complex -> cytochrome c2 -> P870 similarities between Type II and complex III: Complex III cytochrome oxidoreducatse vs pheophytine-quinone type II in bacteria... - Both use a Q cycle! QbH2 for bacteria, QH2 for ETC - QUINONE for bacterias versus ubiquinone for ETC - Both pump 4 protons to P side - Both pass e- to cytochrome C (c2 in bacteria)

Oxygenase activity of Rubsico! - what is a competitor - produces what

O2 is a competitor with CO2 for rubsico intermediate enediol nuclephilic reactive species, and would result in 2-phosphoglycolate (a two carbon moelecule) forming instead of (2) 3-phosphoglcyerates... 2-phosphoglycerate is metabolically useless and salvaging an extra carbon requires wasted energy.

Regulation of Ribonucleotide reductase which convetrs NTPs to dNTPS

ONE ENZYME manages the entire pool of NTPs and dNTPs!!!!! - TWO alpha subunits bind regulators or effector site Regulatory sites: 1. Primary regulatory site: the on/off switch based on relative amounts of ATP and dATP - ATP activates the enzyme! - dATP deactivates (alread have product don't make more, feedback inhibition) 2. Substrate specificity site: Balances relative amounts of specific dNTPs!!! - dATP bound -> enzyme prefers reduction of UDP/CDP (lose an O means reduced) - dGTP bound -> enzyme prefers reduction of ADP - dTTP bound -> enzyme prefers reduction of GDP ---dNTPs steer binding of complimentary nucleotides to maintain proper ratios of the different nucleotides In image, shows two alpha subunits, same exact sites, one side names function other names what binds! Use BOTH (negative) feedback inhibition and POSITIVE allosteric regulation! OVERVIEW: Purines / pyrimidines form in diff paths -> Transphosphorylations to form NTPs -> form dNTP using ribonucleotide reductase

Overview Fatty Acid Synthesis

OVERVIEW: One cycle - 2 reduction step = 2 lost electron carriers NADPH - 1 CO2 removed - 4 e- and 4 H+ are added each round - Malonyl-CoA is the compound added in each step!!! - NOTICE... the malonyl-CoA is added proximally!!!! it attacks the carbonyl carbon and remains part of ACP, then it will be transferred down the enzyme and be transferred to -SH of KS domain and the ACP will bind a new malonyl-coA to again attack carbonyl and add at proximal end! Release requires HYDRATION STEP so the total H2O so for 7 rounds to form 16 carbon palmitate, there are 7 dehydrations (7 H2O - 1 H2O needed to release!) so 6 H2O

Lecture 21 Chapter 20 Part 2 CO2 Assimilation overview - occurs where - key intermediate? (5c) - produces... - NET... (C and anabolic cul) ---- Three stages of the calvin cycle (enzymes) 1. CO2 fix 2. 3-phosph red 3. regen Overall reaction

Occurs in the stroma of chloroplasts using CALVIN cycle - Ribulose 1,5-bisphophate is a key intermediate and constantly regenerated using ATP energy - Produce 3-phosphoglycerate, Dihydroxyacetone phosphate (DHAP), and glyceraldehyde 3-phosphate (G3P/GAP) - NET = reduce CO2 using NADPH that came from photosynthesis -------------------- 1. CO2 Fixation: rubisco (3) ribulose 1,5-bisphosphate + 3 CO2 -> (6) 3-phosphoglycerate in equilibrium with DHAP 2. 3-phosphoglycerate is reduced to glyceraldehyde 3-phosphate using ATP and NADPH from photophosphorylation Enzyme: PGK (phosphoglycerate kinase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 3. Regeneration of ribulose 1,5-bisphosphate - complicated series of reactions involving transketolase (2 c) and transaldolase (3 c) 15 carbons + 3 carbons -> 6 3-carbons -> 6 3-carbons -> 3 5-carbons and a 3-carbon (GAP) overall: 3 CO2 + 6 NADPH + 5 H2O + 9 ATP -> G3P + ADP + 9 Pi + 6 NADP+ + 2 H+

Previous notecard, option B of #3, salvage polar headgroup pathway by chemical modification of diacylglycerol molecule ex serine->enethanolamine Ethanolamin->choline

Once head group is attached, simple chemical transformation b/w head groups can occur! phosphatidylserine->phosphatidylenethanolamine +CO2 PS decaboxyalse phosphatidylenethanolamine + 3 AdoMet -> phosphatidylcholine + 3 AdoHet (AdoMet is a methyl carrier!)

Steps of multi-enzyme complex turning electron carriers into electrons providing potential: florida 1. NADH Dehydrogenase aka NADH:ubiquinone oxidoreductase - conserved channels, sidedness of IM/matrix - FMN - 2 keys - how are protons transferred (proton wire) Overall Rxn

One of the largest macromolecular assemblies in the mammalian cell - four conserved proton channels that transfer protons from the N-Side (negative, the matrix where electron carriers generated) to the P-side (mitochondrial matrix, where protons + charge are pumped) - over 40 diff polypeptides chains, encoded by both nuclear and mitochondrial genes 1. Prosthetic group FMN flavin mononucleotide accepts two electrons from NADH (NADH binds matrix side) 2. Electrons on FMN transfer them down Fe-S centers and pass them one at a time toward the ubiquinone binding site 3. Ubiquinone accepts electrons and becomes reduced to ubiquinol 4. 4 H+ pumped N-side to P-side (IMM) How protons are transferred: - Protons transported by proton wires - series of aa undergo protonation and deprotonation to get a net transfer of a proton from one side of a membrane to another KEYS: A. Pass electrons from NADH -> QH2 B. 4 Protons are pumped in this step, and 2 H+ laterally diffuse with Q as QH2 Overall rxn: NADH + Q + 5 H+ (n-side) <-> NAD+ + QH2 + 4 H+ (P-side) - 5 H+ since NADH + H+ used.

Step by step water-splitting complex donates single electrons to photosystem II but releases a single O2 after four electrons are freed from TWO water explaining the higher plant reaction centers overall equation and flow from H2O to NADPH - X electrons to NADPH per X photons

Overall: 2 H2O -> O2 + 4 H+ + 4 e- 1st exciton, 2nd exciton, 3rd exciton, 4th exciton all needed to strip an electron off of an MnCaO5 cluster by tyrosine, which then gives it to the P680 reaction center... ONLY when the cluster is +4 charge will 2 H2O be oxidized to refuel the electrons and produce 4 H+ all at once. IMAGE IS WRONG: Not proton each step, just four resulting in the end. ------------------ 2 H2O + 2 NADP+ + 8 photons -> O2 + 2 NADPH + 2 H+ --> 4 e- are needed to make 2 NADPH, 4 photons are needed to make one NADPH --> For every 2 photons absorbed, 1 electron is transferred to NADPH You make 1/2 of an O2 molecule with each NADPH made. We have to double the equation to turn 2 H2O into 1 O2. We end up doubling everything else in the process (4 total electrons going to 2 total NADP+). Each electron has to pass through both photosystems to go from H2O to the final NADP+ acceptor, so you need 2 photons per 1 electron, requiring 8 photons total to get 4 electrons to 2 NADP+.

Oxidative phosphorylation versus photophosphorylation: 1. where e- come from and what used to do 2. Ultimate e- acceptor 3. ATP both... provide most ATP in biology! Key: Compare/contrast

Oxidative Phosphorylation: 1. Electrons come from NADH and FADH2... used to oxidize O2 into H2O 2. Ultimate electron acceptor = O2 3. Generates ATP for the cell, couple proton flow to ADP phosphorylation Photophosphorylation: 1. Electrons come from H2O using light energy... used to reduce NADP+ 2. Ultimate electron acceptor is NADPH 3. Generates ATP for the cell, couple proton flow to ADP phosphorylation

transdeamin... Ammonia Lifecycle... collect in glutamate via ______ and then release via _________ - enzyme - purp of oxid deam - either e- carr - exit as - takes place where? - the "interesection ...)

Oxidative deamination: remove ammonia from glutamate - NADP+ or NAD+ can be electron acceptors - Ammonia that comes off will exit via Urea Glutamate + NAD(P)+ <-> intermediate + H2O + NAD(P)H <-> alpha-ketoglutarate + NH4+ - Glutamate dehydrogenase enzyme ------------ Ammonia Lifecycle... collect ammonia using glutamate via transamination and then release it via oxidative deamination -> Transdeamination! = complete pathway for capture then excretion of ammonia (transamination + oxidative deamination) Takes place in the mitochondria!!!!! The intersection of carbon and nitrogen metabolism: Ammonia will be sent to urea cycle, but the alpha-ketoglutarate generated will be sent to the TCA cycle

Lecture 20: oxidative phosphorylation Overview oxidative phosphorylation vs photophosphorylation 1. where take palce 2. where e- come from 3. uses the ____ to work Both: 1. utilize e- flow 2. energy is converted from oxidation to Y building up the 3. ATP created by 4. The ultimate electron acceptor is...

Oxidative phosphorylation: 1. In the mitochondria 2. Electrons come from carriers (NADH, FADH2) from catabolism of carbs, lipids, and amino acids 3. Electron-transport chain used to synthesize ATP Photosynthesis: 1. In chloroplasts 2. Electrons come from H2O which is oxidized to O2 using energy from the sun light 3. Photochemical reaction center (photosystems) used with excited electrons BOTH: 1. Utilize electron flow to produce uphill proton transport across a membrane impermeable to protons 2. Free energy of electrons/redox is converted to electrochemical potential building up the proton motive force 3. Flow of protons down the concentration gradient provides free energy for ATP synthesis in mitochondria and chloroplasts-- couple proton flow to ADP phosphorylation. 4. O2 is the ultimate electron acceptor. These two processes provide most of the ATP in biology!

Aminotransferases the actual chemistry of the reaction with pyridoxal phosphate! pint-pong mechanism

PING 1. Amino acid + pyridoxal phosphate internal aldimine <-> Schiff base intermediate 2. Schiff base intermediate <-> Quinonoid intermediate 3. Attack an H+ for rearrangement to form cyclic ring 4. Hydrolysis of schiff base to form alpha-keto acid amd pyridoxamine phosphate PONG: always is alpha-ketoglutarate to glutamate, regenerate the enzyme Exactly opposite of ping! Forms the amino acid and interal aldimine - lysine from enzyme chases off the amino acid product-> primary amine replaces primary amine! The entire reaction includes both the ping and pong. An amino acids gives up an NH3 to PLP (becoming an alpha keto acid), then the other reactant (alpha keto acid) comes in and takes the NH3 to become an amino acid (and completing the overall reaction). The incoming amino acid is oxidized during the "ping" into a keto acid, while in the "pong" the other reactant keto acid comes in and is reduced to an amino acid.

three general classes (energy fav/netru/cost for each?) 1. transaminations: - reaction - cofactor and enzyme - dG - where does it occur? 2. Oxida deam 3. Glutaminase (just covered a couple slides back!)

PLP Alpha-ketoglutarate + any amino acid <-> Glutamate + some alpha-keto acid - enzyme: aminotransferase (diff each amino acid, there are 19 different ones!) - cofactor is pyridoxal phosphate - dG˚' = 0!!! - makes sense since chemically identical bonds form - Occurs in the cytosol Note: Alpha-Keto Acids are REDUCED to amino acid, but net amino acids/alpha keto acids does not change!

Pyridoxal phosphate structure, internal vs external lys aldimine =

PLP is ALWAYS bound to the enzyme, but only sometimes covalently bonded!!!! Internal Aldimine: Attached to a lysine residue of the aminotransferase ON the enzyme, in the active site (covalent). External aldimine: A good electron sink in Ping as it becomes reduced, is bonded to/transferred to the amino acid substrate as an intermediate, on the outside more of the enzyme (non-covalent interactions). Over the course of the reaction, it will bind and unbind the enzyme lysine residue over and over Aldimine = there is a schiff base present!!!! Good electron sink! -- Able to take many reosnance structures due to conjugations

Regulating the two structures: 1. Cyclic flow b/w PSI and Cyt B6f used to maintain a ratio... 2. organization in thylakoid membrane of photosynthetic machinery - issue if not sep. - plants do... - X is unif distr.

PSII absorbs higher energy molecules than PSI, to keep excitons from migrating immediately to PSI, the complexes can be physically separated by distance. --- BYPASS the Cytochrom b6f complex meaning no ATP (or less at least) generated without proton gradient (8 H+ per O2) A. Plants put PSII in grana stacks B. PSI and ATP synthase are unstacked in stromal thylakoids ---- both have access to stroma (NADP+, ADP, Pi, water, etc.) ---- the close association of PSI and PSII is regulated by sunlight and protein phosphorylation, alters distribution in cell! ---- the location of light harvesting complex can switch from grana (if light is too intense and PSI cannot keep up) to stromal thylakoid (give energy to PSI) C. PSII and PSI get LHCs= noncyclic and NADPH production, PSI only = cyclic and ATP production KEY: Determine cyclic or non-cyclic by bypassing the cyt b6f or by phosporylation or sunlight causing stacks (appressed) containing LHCII to separate (nonappressed) as LHC leave PSII to help out PSI. Since LHCII mediates the stacks, the change from appressed to non-appressed is linked to the separation of LHCII from PSII. Cytochrome b6f is uniformly distributed all over

Summary of plant reaction centers... slide 45 ch20P1 is good PSII: CYT B6F: PSI: total H+ moved per O2... size helps to make it... Energy per proton? Per O2? few diff in ATP synthesis... catalyzed by two domains...

PSII: consume 4 e- which 4 photons, oxidizing 2 H2O to O2. Contirbute 4 H+ to P-side lumen. Cyt b6f: Q cycle that pumps 4 H+ to lumen P-side for every pair of electrons using PQH2 PSI: Absorb 4 more photons and recieve 4 electrons which are capable of generating 2 NADPH from 2 H2O... Removes 2 H+ from stromal n-side making it more negative in synthesizing 2 NADPH _________ 2 H2O -> O2 uses FOUR electrons... 2 electrons: 4 protons from cyt b6f, 2 protons from PSII oxidized H2O = 5 FOUR electrons = 12 protons in P-side Total with 12 protons = 200 KJ/mol! = 3 ATP yield per O2 - enough to drive synthesis of ATP! dG˚' per mole of protons = -17 KJ/mol Since the volume of the lumen is so small, it makes the [H+] even greater, making it a 3 unit pH difference = 1,000 fold difference in [H+]!!! _________ ATP synthase is analagous to motochondria, but CF1/CFo are the complexes used. - found on the OUTER surface (not the outer membrane cause that doesn't exist) of the thylakoid membrane! Synthesize ATP in the stroma (matrix like)

Lecture 21 Chapter 20A Photophosphorylation START OF ANABOLIC REACTIONS 2 phases: 1. light de - ligh abs by - saved as .... - X cons Y prod 2. car fix - driven by - what is fixing carbon (whats involved) - production of... equation... overview photphos: - X is oxid to Y, Z is final e acc - how do we get H2O to donate???

Phases of photosynthesis: 1. Light-dependent: Photophosphorylation - Light energy absorbed by pigments like chlorophyll - Energy is eventually conserved as NADPH and ATP - H2O consumed, O2 produced (opp. oxida. phosph) 2. Carbon fixation/Carbon Assimilation - Driven by the products of photosynthesis (NADPH) - NADPH and ATP reduce CO2 = fixing carbon - "New carbon" thats fixed and existing carbon produce starches, sugar, etc. ... CO2 + H2O -> O2 + Carbohydrate Overview photophosphorylation... H2O is OXIDIZED to O2, and NADPH is formed as FINAL ELECTRON ACCEPTOR - electrons flow through series of membrane bound electron carriers, protons are pumped across a membrane to create a gradient (like Complex III oxidative phosphorylation) used for ATP Synthase - H2O is a bad electron DONOR, O2 is a bad electron acceptor! We use energy from light to change this.

Before: Exciton transfer at a rxn center... NOW: three types of rxn centers... 1. Bacterial Type II-- aka ... - found in - electrons pass thru - properties of pheo - 3 key molecules - What is the light harvesting protein? 2. Bacterial Type I 3. Plant Reaction Centers (type II-type 1 in series)

Pheophytin-Quinone Reaction center -- Type II - Found in: Purple Bacteria - Electrons pass through: Pheophytin - pheophytin is a chlorophyll without central Mg2+ Three key molecules in pheopytin-quinone complex: 1. P870 reaction center (wavelength absorbed) 2. Cytochrome bc1 electron transfer complex - similar to complex III in ETC mitochondria which does Q cycle, oxidizes QH2 to form cytochrome C 3. ATP Synthase Light harvesting pigment = Phycobillins - The light harvesting complex is the phycobilisome protein which funnels light energy to the reaction center

NOW: three types of rxn centers... 1. Bacterial Type II 2. Bacterial Type I 3. Plant Reaction Centers (type II-type 1 in series) - number photons absorbed? - PSII and PS1 - Reduces what, cyclic vs non-cyclic - plastocyanin importance - name for bacteria vs plants Slide 32 Ch20P1 shows diagram of plastoquinones and other key molecules in reaction center (PQa -> PQb) BY THE WAY this is callaed a Z diagram

Plant thylakoids (P-side, Fo of ATPase in thylakoid membrane with ATPase in stroma) - two distinct reaction centers! Type 1 and Type II!!! - They are complementary but separate from one another - hundreds of each system are in the thylakoid membranes - TWO photons are absorbed, one by each reaction center! Plastocyanin: moves electrons between PSII and PSI ONE AT A TIME!!! Bacteria: anoxigenic photosynthesis Plants: Oxigenic photosynthesis Mechanism: A. Photosystem II (PSII)--pheophtyin-quinone type! 0. electrons replaced in photosytem II by H2O oxidized to O2 1. P680 excited, reduce E˚' 2. Electrons transferred to Pheophytin 3. Pheophytin hands off to plastoquinone (PQa) 4. Plastoqunione hands off to Second quinone (PQb) 5. Electrons as reduced PQb now enter the Cytochrome b6f complex which creates a proton gradient, and also hands the electrons off to plastocyanin. 6. Plastocyanin hands them off to photosystem I P700!!! B. Photosystem I (PSI)-- Ferredoxin type! 6. Electrons from Plastocyanin enter P700 and are excited. 7. P700 transfers electrons to Ao electron acceptor chlorophyll 8. Ao passes to A1 phylloquinone 9. Phylloquinone passes to iron-sulfur 10. Fe-S passes to ferredoxin --- TWO directions possible 11. a. Non-cyclic: Ferredoxin to ferredoxin:NADPH reductase to generate NADPH b. cyclic: electrons from ferredoxin re-enter cyt B6f complex to generate greater proton gradient. More negative: higher energy electron donor, low energy electron acceptor.

ACP as the shuttle... Phosphopantheteine (what kind of group) (slide 14 is image of structure) ------ Preparative step... charging FAS two things needed bound, catalyzed by what?

Possesses tether proteins which give it the flexibility to swing the intermediates to the subsequent enzymes in the FAS protein! Esterification to SH allows carrying -> Uses 4'-phosphopantetheine, the SAME tether/carrier for CoenzymeA, actually comes from CoA 1. Deliver acetate in the first step or malonate in ALL other steps to the fatty acid synthase Acts as the SHUTTLE for the growing chain to each enzymatic unit! KS has cysteine ingrained, ACP has prosthetic group thiol ------------- Preparative step: Charging FAS-I KS and ACP - 1. Acetyl-CoA is transferred to thiol in cysteine of KS (we don't discuss but it happens) - 2. Malonyl-CoA is transferred to ACP thiol on phosphopantehteine PROSTHETIC group. Catalyzed by MAT subunit! Malonyl/acetyl-CoA ACP transferase

General four step fatty acid synthase I reaction in words prep, steps 1-4 stoich... stoich of ac to mal rxn stoich of sum total - KEY note on h2o - KEY note on ac coa

Prep: Malonyl-CoA attaches to thioester of ACP, Acetyl group to Cys of KS - Activates the acyl group Step 1: Condesnation reaction attaches two carbon from malonyl CoA to Acetyl - The Malonyl bond attacks the acetyl, releasing CO2 in the process -decarboxylation facilitates the reaction - Creates beta-keto intermediate Step 2: 1st reduction of acetoacetyl group to form a hydroxy group from the beta-keto intermediate Step 3: Dehydration of OH on C3 and H on C2 make double bond via elimination reaction - creates TRANS alkene Step 4: 2nd reduction, NADPH reduces double bond to yield saturated alkane Repeat cycle 7 times total to make palmitate ------------- STOICHIOMETRY of Palmitate: 1. 7 Acetyl-CoA's are used and carboxylated to make 7 Malonyl-CoA's using ATP!!! 7 Acetyl-CoA + 7 Bicarbonate (CO2) + 7 ATP -> 7 Malonyl-CoA + 7 ADP + 7 Pi 2. seven cycles of condensation, reduction, dehydration, reduction, using NADPH to reduce the Beta-Keto group and Trans-alkene double bond Acetyl-CoA + 7 Malonyl-CoA + 14 NADPH + 14 H+ -> Palmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O --> One H2O used to release the palmitate chain! --> One Acetyl-CoA attached to KS cys to start rxn, otherwise only malonyl! - ONLY malonyl ever binds ACP 3. OVERALL reaction starting acetyl-CoA to Malonyl-CoA to FA synthesis! (1+2 before) Acetyl-CoA + 7 Malonyl-CoA + 7 ATP + 14 NADPH + 14 H+ -> Palmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O --> ATP used for attaching CO2 to acetyl-coa --> NADPH reduce to form then remove double bond --> H2O released palmitate (next slide talks about TRUE COST of Fatty acid synthesis (add 2-C units)= 3 ATP since 1 for forming Malonyl and 2 for transporting acetyl-CoA into cytosol!)

Amino group catabolism... step 1- get amino groups to liver! 1. lots of en acq 2. lots of en to dispo _______________ is the key intracellular amino group group carrier once in the liver ---------------------- Digestion of dietary proteins to amino a 1. ingest 2. low pH zym 3. neutr 4. try chy cut 5. single aa high pH zym

Protein metabolism produces a lot of Ammonia which is toxic and metabolically expensive... 1. Lots of energy needed to acquire new nitrogen 2.** Lots of energy to dispose of old nitrogen Transfer amines to carrier molecules for safe transport via blood to the LIVER for processing -> Glutamate is the key intracellular amino group group carrier once in the liver -------------------------------------------- Zymogens = protease precusors, actviated by different means! Pepsinogen for example active in low pH stomach to pepsin 1. Ingest protein through diet 2. low pH stomach ACTIVATES zymogens like pepsinogen which are protease precursors (cut up proteins) and it become pepsin 3. Next to neutral pH and neutralization step in small intestine 4. Next trypsin and chymotrypsin cut proteins and larger peptides into smaller peptides in the small intestine 5. Aminopeptidase and carboxypeptidases A and B are more zymogens activated by HIGH pH and becpme active proteases in SI, degrading peptides into amino acids in the smaller intestine. 6. Pass intestinal lumen and transported into blood stream where taken up by liver

Biosynthesis of Arginine - intermediate seen before 1. n-ac... seen before? purpose? 2. reduction to glutam 3. acetyl(seen before) 4.n-actornths 5-7 from urea...

Requires 1 NAD(P)H and 2 ATP - phosphorylation then reduction - ARG can be used to make proline via ornithine in mammals - proceeds through ornithine intermediate seen in the UREA cycle! ---------- Citrate -> alpha-ketoglutatarate -> Glutamate... 1. Glutamate -> N-Acetylglutamate Acetylglutamate synthase - seen in carbamoyl phosphate synthetase regulator in urea cycle! - N-acetyl group prevent spontaneous cyclization seen in proline!!!! 2. N-Acetylglutamate + NAD(P)H + H+ -> N-acetyl-glutamyl-phosphate + NADP+ N-acetylglutamate dehydrogenase 3. N-acetyl-glutamyl-phosphate + Glutamate -> N-Acetylornithine + Alpha-ketoglutarate Aminotransferase 4. N-Acetylornithine + H2O -> Ornithine + CH3COO- N-Acetylornithinase 5. Ornithine + Carbamoyl phosphate -> l-Citrulline + Pi Ornithine carbamoyl-Phosphate transferase 6. Citulline + ATP + Aspartate -> Arginosuccinate + AMP + PPi Arginosuccinate synthetase 7. Arginosuccinate -> Fumarate + Arginine Arginosuccinase

Biosynthesis on Proline in Bacteria... 1. gltyl phs 2. semiald 3. P5C 4. final prod

Requires 2 NADPH/NADH and 1 ATP - phosphorylation then reduction ------------- Citrate -> alpha-ketoglutatarate -> Glutamate... 1. Glutamate + ATP -> Glutamyl Phosphate + ADP Glutamate Kinase 2. Glutamyl-Phosphate + NADPH + H+ -> Glutamate semialdehyde Glutamate dehydrogenase 3. Glutamate semialdehyde -> Proline-5-carboxylate NON-ENZYMATIC!!! Spontaneous 4. Proline-5-carboxylate + NAD(P)H + H+ -> Proline Proline Carboxylate Reductase

Stage 1 Carbon fixation enzyme dependent on what prosthetic group? Catalyzes... name carb part comes because... Danger with oxy ------- TWO FORMS of rubisco... 1. plant/alg/cyan 2. Photosyn bact note: turnover/activity (how to overcome)

Ribulose 1,5-bisphosphate carboxylase/oxygenase Rubisco: Most abundant enzyme on the planet by mass, in all plants - Mg2+ dependent! --> is a - Catalyzes rib 1,5-bp -> (2) 3-phosphoglycerate - highly regulated (rubisco) - 4˚ quatenary structure ... this enzyme is key to life Carboxylase function: Incorporates inorganic CO2 into organic ribulose 1,5-bisphosphate to form a new C-C bond and 6 carbon intermediate -> Will then cleave the 6-carbon intermediate into (2) 3-phosphoglyerates Danger with O2: - Rubisco can react with O2 in an inefficient side reaction consuming energy without producing anything ------------------ Form 1: Plants, algae, cyanobacteria - 8 large catalytic subunits (encoded by plastid genome) + 8 small subunits (encoded by nucleus) Form 2: Photosynthetic bacteria only - 2 catalytic subunits only, resemble plant subunits NOTE****** Super low turnover number of 3 s^-1 at 25˚ celsius. - Overcome slow turnover by having TONS of enzyme around

End amino acids, start nucleotides! roles of nucletoides? Two synthetic paths 1. de - begin w precur - key prec 5'ph, 1' ppi a. pur (ribo) - carbon sources (with image) b. pyrimi (oro)

Roles of nucleotides: - DNA/RNA (genetic storage), Adenine and FLavin (electron carriers), A and G in ATP/GTP for donating phosphoryl/energy. cNTP (2˚ messengers), Sugar intermediates in synthetic rxns (today) TWO synthetic paths for nucleotides: 1. De Novo Pathway: - Begins with metabolic precursors (PRPP, CO2, amino acids, ribose 5-phosphate, and NH3-) ... NOT with free bases - Key starter is PRPP - phosphorylated ribose precursor 5'-phosphoribosyl-1'-pyrophosphate a. Purine -> made from small precusors and attach to ribose - each carbon from different source... 2 from glycine, 2 formate. 2 glutamine, 1 CO2, 1 Aspartate b. Pyrimidine -> Made from Orotate, then attached to ribose phosphate! 2. Salvage pathways (not covered) - recycle free bases and nucleosides from catabolic pathways! (re-use large building blocks)

End on a new mini topic: Single carbon transfer agents (very important in amino acid metabolism) 1. biotin 2. tetrahydrofolate 3. AdoMet - transfers - synthesized from - regeneration uses - key note on its structure that is unique

S-AdenosylMethionine (SAM) is the preffered cofactor for methyl transfer in biological reactions! - It is 1000 times more reactive than THF methyl group It is synthesized from ATP and methionine condensation Regernated using N5-Methyl THF UNIQUE: The methyl group attaches directly to the sulfur of the 5' carbon adenosine/end of the methionine skeleton! - When it transfers methyl, this holding method makes it have to be fully degraded and regenerated to actually release the CO2 Appears phosphatidylenethanolamine-choline modifying polar head groups on fatty acids (diacylglerol)

Q Cycle in Complex 3 step 3... both steps:

STEP 1: form cytochrome C reduced and semiquinone 1. On the P side (matrix) of membrane, two QH2 molecules (2 protons) are oxidized to Q, releasing 2 H+ into the intermembrane space (IMS) 2. QH2 also release 2 electrons, one will go thru Fe-S centers and heme up to cytochrome C to reduce it, the second will travel down heme groups towards N side where an oxidized Q will bind, and the single electron will turn Q into a semiquinone! Step 2: 3. On the P side of the membrane, another QH2 is oxidized to Q release 2 protons to the IMS (net 4 now) andone electron goes up to cytochrome C reduced and the semiquinone receives its second e- to form QH2 to be used in a subsequent rxn, and 2 cytochrome C, and 4 H+ pumped out. NET: (sep rxns in image) QH2 + 2 Cyt c1 (oxid) + 2 H+(n-side) -> Q + 2 Cyt c1 (red) + 4 H+ (P-side)

Lec 22- Chapter 22 AA synthesis! + wrap up Overview... - nit cycle is Nitrogen cycle components 1. fixat ... atm -> amm is... (oxid state) 2. nitrif ... ammon-> nitri (oxid sta) -> others 3. assim -> N lifecycle in plants/bact 4. denitrifi ... nit ox (oxid st) -> make N2 - ultim e- acc -------- Symbiotic rlntship w soy beans!

Significant amount of nitrogen is required by organisms for nucleotide and amino acid synthesis... - biologically useful nitrogen is relatively rare/not naturally occurring (Non-reduced N2 is common) - Nitrogen Cycle: provides nitrogen in useful forms and allows organisms to conserve/salvage/recycle nitrogen! The Nitrogen Cycle: Chemical transformations maintain balance between N2 and biologically useful forms of Nitrogen. 1. Nitrogen fixation: N2 (0) in atmostphere -> NH4+ (-3) Ammonia - Reduction by bacteria/archaea! --> ammonia can be used by animals to form other reduced nitrogen-carbon compounds! 2. Nitrification: NH4+ -> NO2- nitrite (+3) - Plants or other nitrifying bacteria and archaea oxidize ammonia to NO2- -> Nitrite oxidized to NO3- nitrate (+5) (denitrify or nitrify to convert between the two) 3. Assimilation: Plants and microogranisms reduce NO2- and NO3- back to ammonia or NH3 via nitrite reductases and nitrate reductases -> plants die and release NH3 into soil, bacteria will re-nitrify to nitrite/nitrate 4. Denitrifcation: Nitrite/nitrate converted back to N2 under anaerobic conditions, NO3- is ultimate electron acceptor, NOT O2. --------------- Bacteria are essential with providing biological nitrogen sources to plants!!!! Soy exampls: - Biologically useful nitrogen rare, so we need bacteria to make it. Legumes have root nodules littered with bacteria that fix nitrogen! - This is an intensely energetic cost, but they then have supplies of nitrogen for biological synthesis! Explains why soy is such a common crop/they have a evolutionarily competitive advantage.

FINAL course overview continued: ch 22 Slide 55: Amino acid fates 1. deliver to .. for synthesis 2. other tiss, exp 3. synth other 4. if high, use to make .. and remove and put into cycle Slide 57: Lipid Metabolic fates 1. prim oxid fuel for... - how it gives energy, what precursor? where it goes - turn to X to cross BBB 2. conver to storage 3. what activates stored culs

Slide 55: Amino acid fates 1. Hepatocyte delivery for protein synthesis! - liver does lots of syntehsis since protein turnover rate is high (T1/2 = days) - Liver is site of most blood plasma protein synthesis other than antibodies 2. Protein synthesis in other tissues - amino acids exported from the liver into the blood (glutamine/alanine) for other organs 3. Synthesis of other nitrogenous molecules - nucleotides, hormones, cofactors, secondary messengers 4. If protein/nucleic acid synthesis are stable accompanied by a rise in glutamate/glutamine levels... - Deamination and catabolism of amino acids to Acetyl-CoA, a kreb's cycle intermediate! Acetyl-CoA can be: - convert to glucose/glycogen via gluconeogenesis - oxidzied to help for ATP - converted into lipids - convert resulting ammonia to Urea - the significant loss of N must be recovered through diet! Slide 57: Lipid Metabolic fates 1. Fatty acids are the PRIMARY oxidative fuel of the liver - acetyl-CoA oxidized via the CAC! - provides e- carriers for ATP synthesis using ATP synthase/ETC - energy for nervous system - Acetyl-CoA convert to ketone bodies that circulate blood and cross blood/brain barrier. - cholesterol biosynthesis! 2. Convert to phospholipids and triacylglycerols, carried by plasma lipoproteins to heart, skeletal muscles as fuel 3. Store in adipose tissue - lipases will release fatty acids in response to metabolical needs

FINAL course overview: ch 22 Side 51: Regulation w mass action/acc con Slide 53: Liver metabolism of sugars Levels of control 1. transporter... functions by 2. processing... makes, which functions to - all others converted to this Slide 54: Gluc stored in liver, made into G 6-P now we mobilize it... 6 fates 1. deph 2. store 3. energy 4. anabol/nucleo 5. rltn to ac coa for FA/chol

Slides 49-50 are ALL paths we have learned! Side 51: Regulation w mass action Acceptor Control: Concentrations of ADP/NAD+ (reactant/product availability) control rates of glycolysis and krebs cycle! Slide 53: Liver metabolism of sugars Levels of control: 1. Liver glucose transporter - GLUT2 = passive glucose transoprter, therefore the blood glucose is DETERMINED by the glucose levels in the lvier - absorb or release glucose in response to high/low levels of glucose 2. Liver glucose processing: phosphorylation by glucokinase -> glucose 6-phosphate - Glucose 6-phoshate LOCKS the glucose variant in the liver, but increases concentration of glucose overall in the cell so that glucose can flow out into the blood! - Mannose, fructose, galactose all converted to G 6-P Slide 54: 5 fates of G 6-P 1. Dephosphorylation to form glucose, export to blood 2. Conversion of G 6-P to glycogen for storage 3. Oxidation of G 6-P in glycolysis, krebs cycle (oxidative respiration), or fermentation 4. PPP (oxidative path) makes NADPH and ribose phosphates 5. Glucose-> Acetyl-CoA from oxidative respiration can be used for fatty acid synthesis or cholesterol synthesis

Stage 4 Conversion of Squalene to produce chole core

Squalene fuses to form the four core rings! An O2 atom will be added to the end of the chain! -> forms an epoxide -> catalyzed by mixed function oxidase (squalene monooxygenase -> other oxygen is reduced by NADPH to produce water Lanesterol is the cholesterol CORE -> 19 more rxns yeild cholesterol

Urea Cycle: step ZERO: forming carb phos and occurs in _____ - rxn... -enzyme - ATP requirement - occurs where track amino acids...

Step 0: Formation of carbamoyl phosphate in mitochondria! - bicarbonate from respiration ATP + Bicarbonate -> ADP -> carbonic-phosphoric acid anhydride + NH3+ -> Carbamate + ATP -> Carbamoyl phosphate + ADP -> enzyme: Carbamoyl-phosphate synthetase I -> TWO required ATP's are needed to process ammonia!!! Carbamoyl-phosphate enters urea cycle! OCCURS IN THE MITOCHONDRIA where oxidative deamination is! ----------------------- Glutamate in cytosol transported to mitochondria -> stirp ammonia and put in urea cycle -> an aspartate is added into urea -> step 3 form an arginine -> Urea

Urea Cycle: FOUR ENZYMES step 1: form citrulline from.... step 2: arginosuc synth - burns how many atp - how many aminos present Step 3: arginosucc Step 4: argina - regenerate, which transp where - Urea made up of two N but from where? --- OVERALL ENERGY and equation! - number ATP, another energy product generated?

Step 1: Ornithine transcarbamoylase Ornithine + Carbamoyl-phosphate -> Citrulline + Pi -------------- Step 2: Agrinosuccinate synthetase - enzyme causes formation of activated intermediate, and then catalyzes condenstion of intermediate and asparatate Citrulline + ATP -> [Citrullyl-AMP intermediate] + Aspartate -> Arginosuccinate + AMP - Burns two more ATP equivalents! (ATP-> AMP) - TWO amino groups are now in the molecule! -------------- Step 3: Arginosuccinase Arginosuccinate -> fumarate + Arginine --------------- Step 4: Arginase (hydrolysis reaction) Arginine + H2O -> Urea + Ornithine - ornithine can cross mitochondrial membrane to undergo step 1 back again in mitochondria - Urea: one nitrogen from carbomyl-phosphate, one from aspartate to exit body! ---------------- Overall equation/Energy: three ATP consumed in equation, but FOUR equivalents of ATP are consumed! 2 NH4+ + HCO3- + 3 ATP + H2O -> Urea + 2 ADP + 4 Pi + AMP + 2 H+ -> Step 0 with bicarbonate -> carbamoyl phosphate = 2 ATP -> step 2 Citrulline -> arginosccunitate (ATP->AMP) = 2 ATP -> Fumarate generated from urea cycle for Krebs to generate energy

Stage 2: 3Pg to GAP similar to... Use X instead of Y enzymes.. driven forward by how many set per round? 1. form 13biphos 2. form gap key notes on 1 and 2: (what makes it move, spont or no) How can it be used?

Step 2 in calvin cycle: Reduce 3-phosphoglycerate to Glyceraldehyde 3-phosphate 3-Pg + ATP+ NADPH -> GAP + ADP + NADP+ + Pi - requires products of photosynthesis - Follows payoff phase of glycolysis step 1 and 2 (6/7)! Except it uses NADPH instead of NADH - Use GAPDH and PGK - driven forward by high NADPH and ATP from photophosphorylation - 6 sets = 1 round of calvin cycle 1. 3-phosphoglycerate + ATP -> 1,3-bisphosphoglycerate + ADP enzyme: 3-phoshoglycerate kinase 2. 1,3 biphosphoglycerate + NADPH + H+-> Glyceraldehyde 3-phosphate + Pi + NADP+ Notes... 1. This reaction has a dG of -18.5 to favor producing 3-phosphoglycerate... it can be run in reverse due to the extremely large accumulation of ATP from photophosphorylation 2. NADPH is e- donor not NADH, and the reaction is dG = 6.3 so actually favors running here in the reverse. Driven by high concentration of NADPH __________________ GAP can 1. Can stay in thylakoid (chloroplast) and be made into starch 2. DHAP exit via transporter and be made into sucrose and undergo glycolysis in cytosol

More in depth of de novo pathway -> Purines! multienz produces _______ which is ... (stability/first cmpd with these 2 struc) cost for generating IMP A. IMP -> AMP - generate/use what for energy, source of N B. IMP -> GMP - generate/use what for energy, source of N KEYS: 1. AMP generation uses versus GMP uses 2. Source of Nitrogen is different! 3. GMP production generates, AMP production generates a

Strategy for purines: STARTS with a ring! 7 multi-enzyme complex work in 11 steps to combine precursor molecules using glutamine + PRPP + Gly + ATP, etc. to produce Inosinate (IMP) - Inosinate = key UNSTABLE intermediate which is first compound with purine and ribose ring! - Start here with ribose donated from PRPP!!!! Same with pyrimidine! COST for this step: 5 ATPs Two Pathways for GMP/AMP: late stages 1. AMP: A. Inositate (IMP) + GTP + Aspartate -> adenylosuccinate + GDP adenylosuccinate synthetase B. adenylosuccinate -> Fumarate + Adenylate (AMP) adenylosuccinate lyase 2. GMP: A. Inositate (IMP) + H20 + NAD+ -> NADH + Xanthylate (XMP) IMP dehydrogenase B. XMP + Glutamine + ATP -> Guanylate (GMP) + AMP + PPi + glutamate KEYS: 1. AMP generation uses GTP -> GDP + Pi versus GMP uses ATP -> AMP + PPi! 2. Source of Nitrogen is different! (asp for AMP, Gln for GMP) 3. GMP production generates an NADH, AMP production generates a fumarate!

1. NADH DH 2. Succinate dehydrogenase- complex II

Succinate dehydrogenase: succinate <-> fumarate - Does NOT pump protons! 1. FAD accepts two electrons from succinate as part of the citric acid cycle - only CAC membrane bound enzyme 2. Electrons are passed one at a time via iron-sulfur centers to ubiquinone which becomes reduced to ubiquinol QH2 Involves 4 proteins and 2 prosthetic groups

End on a new mini topic: Single carbon transfer agents (very important in amino acid metabolism) 1 biotin 2 THF - diff ox - essen vit - used in - key on its use 3.

THF - tetrahydrofolate: - Most versatile-- CH3, CH2OH, and CHO -> Carries different oxidation states of carbon! Forms from Folate, an essential vitamin... prescribed during pregnancy to prevent birth defects! Used in variety of reactions like aa catabolism and nucleotide synthesis! Carbon comes from serine, has a N5 and N10 KEY: Tetrahydrofolate is capable of carrying a CO2 on it, and will be reduced by NADPH which causes it to take new conformations to be contributed to other metabolic processes. Seen in production of amino acids! Serine to glycine

Cholesterol Synthesis STAGE 2: Mavelonate -> Isoprene X ATP? How many rxn steps for the X Two activated w equal energy? PPi end prod central role of isoprenes on slide 50!

THREE ATP are required to transfer a phosphate each in a stepwise manner of 3 rxns to mavelonate. FINAL step= Decarboxylation and hydrolysis of one Pi group-> leads to the 5 carbon diphosphorylated intermediate isoprene... A. D3- Isopentyl pyrophosphate (IPP) B. deimethylallylpyrophosphate (DMAPP) A and B can easily interconvert!!! Both are activated forms of isoprenes, both are pyrophosphates and 5C Slide 50 shows isoprenes as KEY intermediate to MANy other paths... Isoprene -> cholesterol -> vit D, bile acid, or steroid hormones Isoprene -> vit A, E, K, or carotenoids, plant hormones, e- carriers isoprene, rubber, etc.

Light harvesting complexes: (LHCs) what they are (pro pig) - assoc w chl - how can energy be transferred? - solid state?

The LHC is how light can be stored as energy! Light harvesting complex: The combination of a binding protein and its pigment (such as chlorophyll) - Chlorophylls are ALWAYS associated with binding proteins which stabilize them in 3-d space NEVER free floating - DO NOT actually donate any electrons, only allow for exciton transfer SOLID STATE: The conformation where binding protein, pigment, and membrane are all in close contact for optimal and efficient energy transfer from photon to reaction center - Energy transfer requires contact between the pigment, membrane, and binding proteins! -> Membrane associated examples: LHCII 7 chlorophyll a 4 chlorophyll b 2 lutein

Cholesterol health deficiency -- Smith-Lemli-Opitz Syndrome disease itself rxn issue consesqu solution guess vs reality

The disease: Autosomal recessive mutation: diseases is caused by mutation in FINAL STEP enzyme of cholesterol synthesis: 7-dehydrocholesterol reductase ... inhibits function! - Build up of 7-dehydrocholesterol, plus 15-27% reduced yield of cholesterol Reaction: 7-dehydrocholesterol + NADPH -> cholesterol 7-dehydrocholesterol reductase Consequences: Physical- many (lung/brain abnormal) Behavioral: many (autism!) Solution proposition: Not enough cholesterol in the body! GUESS? - treat with increased intake of cholesterol wrong Real solution: Treat with inhibitor of HMG CoA reductase! Cholesterol synthesis intermediates are toxic, so inhibition of the first committed step of cholesterol synthesis (stage 1 to form mavelonate) prevents these intermediates from building up! - symptoms not due to lack cholesterol but instead accumulation of toxic intermediates

Standard reduction potentials (E'˚)

The higher the number gets the electrons! More positive E'˚ means more spontaneous (-dG) E = electron affinity, greater number = more affinity H2O is higher than cytochrome, higher than NADH etc. Each electron transfer step has the ability to do work in the form of proton transport! Eq 1: dE'˚ = E˚' (acceptor) - E˚' (donor) Eq 2: dG˚' = -nF(dE˚') ***n = number of electrons **** DO NOT multiply dE but the number you multiply the 1/2 rxn by to get same number electrons, but do flip sign! Equation 1 measures electron affinity, equation 2 measures free energy available to power the proton pump

Carbon Skeletons feeding the citric acid cycle....

The liver may receive way more amino acids than it needs to function properly/more energy than required... - Carbon skeletons will be exported for other tissues or stored. All Skeletons have their own unique paths due to different chemical structures! Most are both keto and gluconeogenic! Blue in diagram: KETOGENIC - Yielding acetyl-CoA (or acetoactetyl-CoA), which can be used to generate ketone bodies Red in diagram: GLUCONEOGENIC: Capable of conversion to glucose, used as intermediates in krebs cycle Leucine: The ONLY completely ketogenic amino acid, ALL carbons go to ketone bodies generation... - Important because this is one of the most common amino acids, and so high-protein diets dictates we will receive large amounts of KETONE body synthesis! KETOGENIC cannot make glucose since you need >2 carbons to make glucose so acetyl-coa cannot count!

Regulating the two structures: 1. Cyclic flow b/w PSI and Cyt B6f used to maintain a ratio... 2. organization in thylakoid

The non-cyclic flow generates NADPH and also consumes protons contributing to Proton gradient... However, if we want to tune the NADPH/ATP ratio, the cell can do more cyclic flow which ONLY creates a proton gradient without generating NADPH, so will increase ATP relative NADPH. --> Change where light harvesting complexes are found, put them more towards PSI (Stromal thylakoid) for NADPH and linear reaction. Put them more towards PSII (Grana) for ATP synthesis and cyclic cycle. No O2 is made, since no H2O needed to replace electrons. Cyclic photosynthesis = generate excess ATP Non-cyclic = anabolism and NADPH/ATP made

4 components of the electron transport chain: 1./2 complex 1 and 2 cat. transfer to 3. carry e' from Q to C 4. trans e C -> O Chemiosmotic energy coupling requires membranes explain why and where takes place in bact, mit, chlor 1. electr down H up 2. H down adp up

The protein components of the mitochondrial respiratory chain: I: NADH dehydrogenase - prosthetic: FMN, Fe-S - Catalyze transfer of electrons from NADH to Coenzyme Q/uniquinone II: Succinate Dehydrogenase - prosthetic: FAD, Fe-S - catalyze the transfer of electrons from succinate (FADH2) to CoQ/Ubiquinone III: Ubiquinone: cytochrome c oxidoreductase (or cytochrome c) - prosthetic: heme, Fe-S (or just heme) - Carries electrons from reduced ubiquinone to cytochrome C IV: Cytochrome oxidase - prosthetic: Hemes; Cua, Cub - transfers electrons from Cytochrome C to O2 ---------------- Proton gradient to generate ATP synthesis can be stably established across a membrane that is impermeable to ions! - plasma membrane of bacteria - inner membrane of mitochondria - Thylakoid membrane in chloroplasts 1. Membrane contains proteins that couple "downhill" flow of electrons in the electron transport chain (ETC) with the UPHILL flow of protons across membrane 2. Membrane contains then a protein coupling downhill flow of protons to phosphorylation of ADP

Why does rotation cause ATP synthesis (it leaves enzyme alpha/beta domain even though it's tightly bound)? process number of protons per c...

The three alpha/beta domains forming the hexamer all will associate with the GAMMA stalk one at a time. When the stalk interacts with the beta catalytic unit on the F1 domain it forces the ATP to disassociate. Now as the protons pass the a- Fo subunit it causes the carousel c subunit to rotate which rotates stalk and keeps pushing off ATP at new beta catalytic subunits. Protons enter matrix, this decreases the established electrochemical gradient! Protons per c-subunits: EVERY hexamer alpha-beta complex provides THREE ATP per turn... - If there are 9 c-subunits, 9/3 means one turn generates 3 ATP (always) but requires 3 H+ per ATP - 12 c-subunits means 12/3 ATP generated = 4 H+ per ATP ... # c-subunits / 3 ATP = X proton transfers needed per ATP

Stage 3 cholesterol synth Isoprenes -> squalene what fuels the rxn? which req NADPH? h2h h2t

This energy reaction utilizes pyrophosphates energy to occur! (PPi byproduct) Sequential condensation reactions releasing PPi! -> Head to head requires input of NADPH - Head to tail do not use energy 1. Geranyl pyrophosphate (GPP) is first intermediate with two isoprenes joining head to tail 2. GPP + IPP = farnesyl pyrophosphate is next intermediate using head to tail 3. add farnesyl pyrophosphates together head to head (use NADPH)

Converting mononucleotides to tri... mono diphos

Transphosphorylations: 1. ATP + AMP -> 2 ADP or ATP + NMP -> NDP + NDP Nucleoside monophosphate kinase, ex: adenylate kinase, guanylate kinase, etc. 2. ATP + NDP -> ADP + NTP Nucleoside diphosphate kinase

NADH SHUTTLES... shuttle vs transporter what is the issue? ... solution? 1. Malate-Aspartate shuttle Number ATP produced with this step?

Transporter: ONE protein or protein complex moving a solute(s) as symporter, antiporter, or uniporter. Shuttle: GROUP of proteins (usually transporters) that function coordinately to move molecules in/out Issue: Some NADH (like that made in glycolysis) is in CYTOSOL... the mitochondrial membrane is impermeable to NADH so needs transporters to reach NADH dehydrogenase complex I in mitochondrial matrix/inner membrane Solution: Move "equivalents" of NADH by reducing other molecules and carbon skeletons that can travel through the membrane. _________ Malate-aspartate shuttle: A) Oxaloacetate is reduced to malate via malate dehydrogenase B) Malate-alpha-ketoglutarate transporter shuttles malate into the matrix N-side C) Malate dehydrogenase oxidizes malate to oxaloacetate, effectivly transporting 2 e- to NADH + H+ for the mitochondrial ETC. D. Oxaloacetate -> aspartate transfer to cytosol -> back to oxaloacetate 32 ATP produced with this step from a glucose

Synthesis of Diacylglycerol (no head group yet... next slide) 1a. same as NADH shuttle 1b. simple kinase glyc 2. form key product (phos) using enzyme Where else this is used? energy track (AMP hint)

Two routes to start: 1A. DHAP + NADH -> Glycerol 3-phopshate+ NAD+ Glycerol 3-phosphate dehydrogenase (from glycolysis or from shuttle of NADH to ETC!) 1B. Glycerol + ATP -> Glycerol 3-phosphate + ADP Glyercol 3-kinase 2. Glycerol 3-phosphate + Fatty-Acyl CoA -> phosphaltidic acid Acyl transferase For step 2... notice there is a side reaction with Fatty Acyl-CoA Synthetase (adenylylation reaction) allowing the acyl to transfer onto the glycerol molecule! Phosphatidic acid is a key intermediate in glycerophospholipid synthesis! The template molecule! ENERGY TRACKING: - 2 fatty acyl-CoA needed, two Acyl-CoA Synthetase 2 ATP-> AMP = four ATP equivalents per diacylglycerol

Uncoupling and not ATP synthesis... thermogenin signalling

Uncoupling where proton gradient and ETC operational but no ATP synthase... the energy is used to generate HEAT! Weight pills use DNP to decrease over energy production and storage of fat, side effect is hyperthermia! Positive: BROWN ADIPOSE FAT tissue-- infants and hibernating animals kept warm by this, protons moved to matrix without generating ATP -- non-shivering thermogenesis Thermogenin Signaling: - Uncoupling protein UCP1 (thermogenin) will uncouple ATP synthase and O2 oxidation, dissipates energy as heat.

NOW: Pyrmidine synthesis - unique that pyriminde does not start w a ... - All made from (2 parent cmpds0 - 1 make ring in form of... then attach to ... - Carbon atoms come from _____ and ______ - what donates the ribose?? - what is the first possible pyrimidine? - what donor helps get other pyrimidines?

Unlike purine, pyrmidine synthesis start with a linear molecule (no ring!) - PRPP donates the ribose!!! PRPP also ribose source in Purines! All pyrimidines are made from UMP and CMP parent compounds. 1. Make pyrimidine ring in form of orotate, then attach it to Ribose 5-phosphate Atoms forming carbons = Aspartate and Carbamoyl Phosphate! MECHANISM: 1. Aspartate + Carbamoyl phosphate -> N-carbamoylaspartate + Pi Aspartate trans-carbamoylase 2. N-carbamoylaspartate -> L-Dihydroorotate + H2O Dihydroorotoase 3. Dihydroorotate + NAD+ -> NADH + Orotate Dihydroorotate dehydrogenase 4. Orotate + PRPP -> orotidylate + PPi orotate phosphoribosyl-transferase - PRPP donates the ribose!!!! 5. orotidylate -> Uritidylate (UMP) + CO2 Orotidylate decarboxylase 6. UMP + 2 ATP -> UTP Kinases 7. UTP + glutamine + ATP-> CTP + ADP + Pi + glutamate KEYS: 1. first possible pyrimidine is UMP, formed from adding ribose 5P via PRPP then decarboxylation 2. Glutamine donor helps turn UTPs into other pyrimidines like CTP!

In the liver: Nitrogen Excretion and The Urea Cycle Step 1: liver transamin (where) Step 2: cross memb Step 3: oxid dea (where) lastly will be what intermed, then shipped where

Urea-> blood -> kidney -> urine Cells within the LIVER: Step 1: Transamination (form glutamate from alpha-ketoglutarate) in the CYTOSOL Step 2: Glutamate crosses mitochondrial membrane via transporter Step 3: Oxidative deamination in mitochondria (glutamate release ammonia for urea cycle, and become alpha-ketoglutarate for TCA) -> intersection of carbon and nitrogen metabolism Ammonia will then be converted into carbamoyl phosphate in the mitochondria to enter urea cycle, where it is transported out after this first step to take place in the cytosol!

Acetate Shuttle: three transporters: all are _____ which one is used in FA synthesis true cost for one 2C making Steps: 1. ac/oxal TCA form X 2. X leaves and enters 3. cit -> ox + ac, where ac goes, where ox goes 4. Ox to mal using 5. a. mal enter via mal/ket anti, do this route a if... b. Mal to pyru, then trans, then conv back, uses/makes... do this route if....

Uses THREE transporters: (all antiporters) 1. Citrate transporter 2. Malate/alpha-ketoglutarate transporter 3. Pyruvate transporter (FA SYNTHESIS ROUTE, consumes 2 ATP total, and makes one NADPH and NADH in cytosol) - true cost of one 2-C addition FA synthesis = 3 ATP due to 1 from Acetyl-CoA -> Malonyl-CoA and 2 for transport Steps: 1. Acetyl-CoA and Oxaloacetate in the mitochondria (TCA cycle) use citrate synthase to form citrate 2. Citrate leaves matrix via citrate transporter into mitochondria 3. Citrate regenerates oxaloacetate and acetyl-CoA using Citrate Lyase at the cost of 1 ATP - Acetyl CoA will be used in FA synthesis 4. Oxaloacetate will be reduced to malate via malate dehydrogenase 5. choices a. Malate can enter via malate/alpha-ketoglutarate ANTIPORTER (alpha-ketoglut absent from pic) to be oxidized to oxaloacetate and produce electrons for the electron transport chain! b. Malate will be converted to pyruvate using MALIC ENZYME, releasing CO2 and generating an NADPH. Pyruvate will enter the cell and pyruvate carboxylase will convert it to oxaloacetate using up an ATP and CO2 Route a: Malate will be used to deliver electron carriers INTO the mitochondria, do this when you already have lots of reduction potential in the cytosol! Route B: Malate converted to pyruvate which makes energy in the cytosol, and uses up energy in the matrix! Use this in times you need to MAKE more reductive potential in the cytosol for anabolism! -> FA synthesis route since we need more NADPH! Uses TWO ATP!!! NET: Oxidize an NADH and make an NAPDH for anabolism!

Light Absorption: vis light is.... wavelengths higher vs lower energy an einstein

Visible light is electromagnetic radiation - 400 nm - 700 nm LOWER wavelength = HIGHER energy (400 > 700) One mole of photons of visible light = 170-300 KJ, more than enough to phosphorylate ADP (req. 30-50 KJ) 1 einstein = 1 mol of photons

Why does rotation of the carousel (c units) cause ATP synthesis? steps (mini rxn eq) enzyme binding... solved issue, new problem, new solution ------ F1 catalysis - subunits/domains 3 conformat. note on catalytic subunit explain graph and image: "Binding-change model"

enz-ADP+Pi -> Enz-ATP -> ATP - bolded = the step where rotational catalysis occurs ATP synthesis is not favorable but the enzyme binds ATP wayyyy tighter than ADP/Pi ATP binding releases so much energy (most stable intermediate, lowest on curve dG) that it counterbalances the cost of making the new bond ADP with Pi in the subunit. Solved one problem: formed ATP from ADP and Pi New problem: ATP is bound tight, how take it off? Solution: Proton gradient drives release of ATP!!! ----------- F1 catalyzes ADP + Pi <-> ATP - heterohexamer (three subunits alpha and three betas) - alpha/beta dimer subunit! Three conformations: each dimer takes on one of the three at any point, always an open, loose, and tight present! 1. Open: empty bound nothing ... "Beta empty" 2. Loose: bound ADP + Pi ... "Beta ADP" 3. tight: catalyze ATP formation and bind ATP product ... "Beta ATP" Image: - notice the lever where it swings it knocks off ATP NOTE: Only the Beta subunit ever binds anything!!!!!! Alpha never does! Beta is the catalytic subunit! GRAPH: 1. typical enzyme just lower dG duble dagger, activation energy 2. The ATP synthase alpha/beta dimer mechanism actually involves many intermediates. Enzyme bind ADP + Pi is favorable, enzyme binding ATP is more favorable so drives the reaction... NOW we have issue of how to get ATP off... this is where the step is coupled to proton flow through Fo.

Exciton Transfer: The photophosphorylation oxidation-reduction reaction efficiency of transfer in center? stored in intermediates? dE / dG? speeds? Steps of exciton trranfser and redox rxn chlor a vs b conclusion on rxn centers

light harvesting chlorophyll in reaction centers EFFICIENTLY transfer energy! Little is lost. - Electron transfer is carefully controlled by precise structural arrangements of interacting molecules = extreme speeds. Essentially no energy is stored in intermediates. dG˚' = -180 KJ/mol and dE˚': .95 V The antenna's show how electrons from light will jump to a higher energy level (exciton), they then can pass energy to a neighboring chlorophyll molecule and they themselves return to lower energy level... this is known as a EXCITON TRANSFER. This occurs until a special pair at chlorophyll a is excited at the reaction center! 1. Absorb photon and excite a pigment 2. Transfer this energy to neighbor (since solid state configuration, other pigments are close and can do so) 3. Repeat until reaction center is reached- chlorophyll a 4. A electron here is donated from an adjacent molecule to the electron acceptor known as the special pair! 5. The special pair/electron acceptor gains a negative charge, but the electron donor chlorophyll a is now a positively charged molecule 6. This creation of charge now requires a neighboring donor to transfer an electron, the donor differs based on the photochemical system!!! The reaction center has chlorophyll a (primary pigment of chloroplast) which collects energy from chlorophyll b (accessory pigment)! conclusion: reaction centers are the source of electrons passed down the transport chain!