Lecture 26 –TCA cycle

Page 766

Figure 21-17bFactors controlling the activity of the PDC. (b) Covalent modification in the eukaryotic complex. Page 781

Control by phosporylation/dephosphorylation Occurs only in eukaryotic complexes The E2 subunit has both a pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase that act to regulate the E1 subunit. Kinase inactivates the E1 subunit. Phosphatase activates the subunit. Ca 2+ is an important secondary messenger, it enhances phosphatase activity.

Page 766

Citric acid cycle: 8 enzymes Oxidize an acetyl group to 2 CO 2 molecules and generates 3 NADH, 1 FADH 2, and 1 GTP. 1.Citrate synthase: catalyzes the condensation of acetyl-CoA and oxaloacetate to yield citrate. 2.Aconitase: isomerizes citrate to the easily oxidized isocitrate. 3.Isocitrate dehydrogenase: oxidizes isocitrate to the  -keto acid oxalosuccinate, coupled to NADH formation. Oxalosuccinate is then decarboxylated to form  -ketoglutarate. (1st NADH and CO 2 ).  -ketoglutarate dehydrogenase: oxidatively decarboxylates  - ketoglutarate to succinyl-CoA. (2nd NADH and CO 2 ). 5.Succinyl-CoA synthetase converts succinyl-CoA to succinate. Forms GTP. 6.Succinate dehydrogenase: catalyzes the oxidation of central single bond of succinate to a trans double bond, yielding fumarate and FADH 2. 7.Fumarase: catalyzes the hydration of the double bond to produce malate. 8.Malate dehydrogenase: reforms OAA by oxidizing 2ndary OH group to ketone (3rd NADH)

Citric acid cycle 3NAD + + FAD + GDP + Pi + acetyl-CoA 3NADH + FADH 2 + GTP + CoA + 2CO 2 3NADH + FADH 2 are oxidized by the electron transport chain and drive ATP synthesis.

Citrate synthase: reaction 1 Catalyzes the condensation of acetyl-CoA and oxaloacetate to form citrate. Oxaloacetate has to bind to the enzyme before acetyl-CoA. Oxaloacetate binds to the enzyme causing a conformational shift that opens the acetyl-CoA binding site. (induced fit) Reaction mechanism is a mixed aldol-Claisen ester condensation (acid-base catalysis). Acetyl forms an enol intermediate. Three important amino acids: His274, Asp375, His320 1.The formation of enolate form of acetyl-CoA is the rate-limiting step. Asp375 acts as a general base to remove a proton from the methyl group of the acetyl-CoA. His 274 is hydrogen bonded to acetyl-CoA. 2.Citryl-CoA is formed in a second acid-base catalyzed reaction step. Acetyl-CoA enolate form attacks oxaloacetate. 3.Citryl-CoA is hydrolyzed to citrate and CoA. Stereospecific reactions (acetate onlly forms citrate’s pro-S carboxymethyl group.

Page 783

Aconitase: reaction 2 Catalyzes the reversible isomerization of citrate and isocitrate with cis-aconitate as an intermediate. Citrate is prochiral so aconitase can distinguish between citrate’s pro-R and pro-S carboxymethyl groups. Has a covalently bound [4Fe-4S] iron-sulfur cluster. Fe a atom coordinates with the OH group of citrate The iron-sulfur cluster does not perform a redox reaction but instead is able to stabilize the ligand-substrate complex. Second stage of the reaction rehydrates cis-aconitate’s double bond in a stereospecific trans addition to form only the 2R,3S isocitrate form.

Page 784

Isocitrate dehydrogenase: reaction 3 Catalyzes the oxidiation of isocitrate to form a- ketoglutarate 1st reaction to produce NADH and CO 2. Activated by AMP and ADP Inhibited by NADH and NADPH Competitively bind to the NAD+ binding site. Requires Mn 2+ or Mg 2+ cofactor. Mechanistically-oxidize to the b-keto acid. 2 forms of the enzyme Mitochondrial form is NAD+ dependant [ADP] E. coli, mitochondrial, cytoplasmic forms NADP+ dependant.

Figure 21-21Probable reaction mechanism of isocitrate dehydrogenase. Page 785

 -ketoglutarte dehydrogenase complex Catalyzes the oxidiation and decarboxylation of  - ketoglutarate to produce succinyl-CoA. Consists of  -ketoglutarte dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase (E3). Mechanistically resembles PDC. 2nd reaction to produce NADH and CO 2. 5 coenzymes (TPP, lipoic acid, CoA, FAD, NAD + ) Product inhibition (Succinyl-CoA), NADH

Reaction 1:  -ketoglutarate dehydrogenase CH 3 P-P-O S N R (+) (-) C - O - C=OC=O CH 2 O CH 3 P-P-O S N R (+) CH 2 C - O - C-OH O CO 2 TPP (ylid form)  -ketoglutarate H+H+ E1 CH 2 C-O-C-O- O C-O-C-O- O

 -hydroxy-  -carboxy- propyl TPP-E1 complex - CH 3 P-P-O R CH 2 C-OH S N + Reaction 2: Dihydrolipoyl transacetylase (E2) S S E2 E1 Lipoamide-E2 H+H+  -hydroxy group carbanion attacks the lipoamide disulfide causing the reduction of the disulfide bond CH 2 C-O-C-O- O

CH 3 P-P-O R CH 2 C-O-H S N + Dihydrolipoyl transacetylase (E2) HS S E2 E1 H+H+ The TPP is eliminated to form succinyl - dihydrolipoamide and regenerate E1 CH 2 C-O-C-O- O  -hydroxy-  -carboxy- propyl TPP-E1 complex

TPP-E1 complex Back to reaction 1 CH 3 P-P-O R CH 2 C S N + Dihydrolipoyl transacetylase (E2) E1 - HS S E2 Succinyl- dilipoamide-E2 O CH 2 C-O-C-O- O

CCH 2 -CH 2 -COO- Reaction 3: Dihydrolipoyl transacetylase (E2) HS E2 Succinyl- dilipoamide-E2 O HS S E2 CoA-SH CoA-S dihydrolipamide-E2 E2 catalyzes the transfer of the succinyl group to CoA via a transesterification reaction where the sulfhydryl group of CoA attacks the acetyl group of the acetyl dilipoamide-E2 complex. + C CH 2 O C-O-C-O- O Succinyl-CoA

Reaction 4: Dihydrolipoyl dehydrogenase (E3) HS E2 E3 is oxidized and catalyzes the oxidation of dihydrolipoamide completing the cycle of E2. + FAD S S E3 oxidized FAD SH S S E2 + E3 reduced

Reaction 5: Dihydrolipoyl dehydrogenase (E3) E3 is oxidized by the enzyme bound FAD which is reduced to FADH 2. This reduces NAD + to produce NADH. S FADH 2 S FAD SH NAD + FAD S S NADH + H + E3 oxidized

Succinyl-CoA Synthetase Hydrolyzes the “high-energy” succinyl-CoA with the coupled synthesis of a “high-energy” nucleoside triphosphate. In mammals, GTP In bacteria and plants, ATP.

Succinyl-CoA Synthetase Mechanistically: Enz-His Enz-His-P Succinyl-P Succinyl-CoA PiCoASH Enz-His-P Succinate Mg ++ GTP GDP

Succinyl-CoA Synthetase Mechanistically: Enz-His Enz-His-P Succinyl-P Succinyl-CoA PiCoASH Enz-His-P Succinate Mg ++ GTP GDP

Figure 21-22aReactions catalyzed by succinyl-CoA synthetase. Formation of succinyl phosphate, a “high-energy” mixed anhydride. Page 787

Figure 21-22bReactions catalyzed by succinyl-CoA synthetase. Formation of phosphoryl–His, a “high-energy” intermediate. Page 787

Figure 21-22cReactions catalyzed by succinyl-CoA synthetase. Transfer of the phosphoryl group to GDP, forming GTP. Page 787

Succinate dehydrogenase Only makes the trans-fumarate. Donates electrons directly into complex II of the respiratory chain (ubiquinone (Q)). If the respiratory chain is inhibited, FAD is unable to accept electrons and TCA cycle stops. Inhibited by OAA, activated by coenzyme Q (part of electron tranport chain).

Figure 21-23Covalent attachment of FAD to a His residue of succinate dehydrogenase. Page 787

Succinate dehydrogenase Electron transport chain Succinate dehydrogenase FADH 2 COO - H-C-H Succinate H-C-H COO - H-C-COO - Fumarate - OOC-C-H FAD

Succinate dehydrogenase Catalyzes the stereospecific dehydrogenation of succinate to fumurate. Enzyme strongly inhibited by malonate (competitive inhibitor). Contains an FAD-electron acceptor. FAD functions to oxidize alkanes to alkenes (vs. NAD+ which oxidizes alcohols to aldehydes and ketones). FAD covalently linked to His from enzyme.

Fumarase (fumarate hydratase) Catalyzes the stereospecific dehydrogenation of succinate to fumurate. Only catalyzes the trans-fumarate Competitively inhibited by maleate (cis double- bond).

Fumarase COO - HO-C-H S-malate H-C-H COO - H-C-COO - Fumarate - OOC-C-H H2OH2O

Malate dehydrogenase Catalyzes the final reaction of the citric acid cycle-regeneration of oxaloacetate. Oxidizes S-malate’s OH group to a ketone in an NAD+ dependent reaction. Produces NADH. COO - HO-C-H S-malate H-C-H COO - Malate dehydrogenase NADH COO - O=C-H Oxaloacetate H-C-H COO - NAD+

Page 766

Total (PDH and TCA) 3NAD + + FAD + GDP + Pi + acetyl-CoA 3NADH + FADH 2 + GTP + CoA + 2CO 2 NAD + + pyruvate + CoANADH + acetyl-CoA + CO 2 PDH TCA Pyruvate 4NAD + FAD GDP + Pi 3CO 2 4NADH FADH 2 GTP 12ATP 2ATP 1ATP NADH DH Complex II Nucleoside diphosphokinase

Figure 21-25Regulation of the citric acid cycle. Page 791

Regulation of citric acid cycle Rate-controlling enzymes: citrate synthase, isocitrate dehydrogenase,  -ketoglutarate dehydrogenase. Regulated by substrate availability, product inhibition and inhibition by other cycle intermediates (generally simpler than glycolysis). Citrate synthase- inhibited by citrate,  -KG, succ-CoA, NADH, activated by OAA and CoASH. Isocitrate dehydrogenase-Requires AMP/ADP Activated by Ca 2+, inhibited by NADPH or NADH  -ketoglutarate dehydrogenase-inhibited by Succ-CoA, NADH, ATP. Activated by Ca 2+ Pyruvate dehydrogenase-inhibited by NADH and acetyl-CoA

Figure 21-26Amphibolic functions of the citric acid cycle. Page 793

Pathways that use citric acid cycle intermediates Reactions that utilize intermediates of TCA cycle are called cataplerotic reactions 1.Gluconeogenesis-in cytosol uses OAA. In the mitochondria uses malate (transported across the membrane). 2.Lipid biosynthesis-requires acetyl-CoA. Transported across the membrane by the breakdown of citrate. ATP + citrate + CoA ADP + Pi + oxaloacetate + acetyl-CoA 3.Amino acid biosynthesis-can use  -ketoglutarate to form glutamic acid in a reductive amination reaction (uses NAD+ or NADP+ depending on enzyme)  -ketoglutarate + NAD(P)H + NH 4 + glutamate + NAD(P) + + H 2 O

Pathways that use citric acid cycle intermediates 3.Amino acid biosynthesis-can also use  -ketoglutarate and oxaloacactate in transamination reactions  -ketoglutarate + alanine glutamate + pyruvate oxaloacetate + alanine aspartate + pyruvate 4.Porphyrin biosynthesis- utilizes succinyl-CoA 5.Complete oxidation of amino acids - amino acids first converted to PE by PEPCK

Pathways that make citric acid cycle intermediates Reactions that replenish intermediates of TCA cycle are called anaplerotic reactions Pyruvate carboxylase- produces oxaloacetate Pyruvate + CO 2 + ATP + H 2 O oxaloacetate + ADP + Pi Degradative pathways generate TCA cycle intermediates 1.Oxidation of odd-chain fatty acids generates succinyl-CoA 2.Ile, Met, Val generate succinyl-CoA 3.Transamination and deamination of amino acids leads to  - ketoglutarate and oxaloacetate.

Each NADH yields ≈ 3ATP Each FADH 2 yields ≈ 2ATP Total yields ≈ 38ATP for each fully oxidized glucose.

Page 851

Glyoxylate cycle The glyoxylate cycle results in the net conversion of two acetyl-CoA to succinate instead of 4 CO 2 in citric acid cycle. Succinate is transferred to mitochondrion where it can be converted to OAA (TCA) Can go to cytosol where it is converted to oxaloacetate for gluconeogenesis. Net reaction 2Ac-CoA + 2NAD + + FAD OAA + 2CoA + 2NADH +FADH 2 + 2H + Plants are able to convert fatty acids to glucose through this pathway

Page 798