Fundamentals of Biochemistry Third Edition Fundamentals of Biochemistry Third Edition Chapter 17 Citric Acid Cycle Chapter 17 Citric Acid Cycle Copyright © 2008 by John Wiley & Sons, Inc. Donald Voet Judith G. Voet Charlotte W. Pratt

The story so far: Glycolysis takes glucose to pyruvate The citric acid cycle = TCA = Krebs’ cycle (Hans Krebs, 1937) = the tricarboxylic acid cycle has as an input the high-energy molecule acetyl- CoA. Pyruvate loses a carbon dioxide and a pair of electrons to make acetyl- CoA. Ultimately, the acetyl group is oxidized to two carbon dioxide molecules, with four pairs of electrons being transferred.

The pyruvate to acetyl-CoA step is considered to be part of the cycle in the text, because all these reactions take place in the mitochondrion (glycolysis took place in the cytosol). The citric acid cycle classically has acetyl-CoA enter during its reaction with oxaloacetate, with oxaloacetate being regenerated at the “conclusion” of the cycle.

Net reaction: 3 NAD + + FAD + GDP + P i + acetyl-CoA + 2 H 2 O  3 NADH + FADH 2 + GTP + 2 CO H +

Pyruvate dehydrogenase is a multi-enzyme complex, which are groups of non-covalently associated enzymes that perform two or more steps in a metabolic pathway. The micrograph shows the symmetry associated with this complex.

Pyruvate dehydrogenase has a lot of helper molecules

Pyruvate dehydrogenase is a multi-enzyme complex, which are groups of non-covalently associated enzymes that perform two or more steps in a metabolic pathway. Its structure consists of three enzymes: E1 (also confusingly named pyruvate dehydrogenase), E2 (dihydrolipoyl transacetylase) and E3 (dihydrolipoyl dehydrogenase).

Pyruvate dehydrogenase is a multi-enzyme complex, which are groups of non-covalently associated enzymes that perform two or more steps in a metabolic pathway. Its structure consists of three enzymes: E1 (also confusingly named pyruvate dehydrogenase), E2 (dihydrolipoyl transacetylase) and E3 (dihydrolipoyl dehydrogenase). 24 cubically-arranged E2 enzymes at the “core” of the complex

Pyruvate dehydrogenase is a multi-enzyme complex, which are groups of non-covalently associated enzymes that perform two or more steps in a metabolic pathway. Its structure consists of three enzymes: E1 (also confusingly named pyruvate dehydrogenase), E2 (dihydrolipoyl transacetylase) and E3 (dihydrolipoyl dehydrogenase). 24 E1 enzymes at the edges of the cube and 12 E3 enzymes at the corners on the “periphery” of the complex

Pyruvate dehydrogenase is a multi-enzyme complex, which are groups of non-covalently associated enzymes that perform two or more steps in a metabolic pathway. Its structure consists of three enzymes: E1 (also confusingly named pyruvate dehydrogenase), E2 (dihydrolipoyl transacetylase) and E3 (dihydrolipoyl dehydrogenase). The E2 and E3 enzymes form dimers that attach the core to the periphery. There are other enyzmes (binding, kinases and phosphatases) that associate with the complex.

Pyruvate dehydrogenase catalyzes the reaction: pyruvate + CoA + NAD +  acetyl-CoA + CO 2 + NADH E2 portion in the periphery Most of E2 in the core

Pyruvate dehydrogenase catalyzes the reaction: pyruvate + CoA + NAD +  acetyl-CoA + CO 2 + NADH The fascinating lipolysyl “swinging arm” that connects the core with the periphery

Pyruvate dehydrogenase catalyzes the reaction: pyruvate + CoA + NAD +  acetyl-CoA + CO 2 + NADH The fascinating lipolysyl “swinging arm” The cyclic disulfide binds to and transfers the acetyl group from E1 (where the pyruvate is decaroxylated) to coenzyme A on E2.

The mechanism of the acetyl transfer: thiamine pyrophosphate (TPP) allows the stabilization of the carbanion that nucleophilically attacks C2 on pyruvate, allowing the decarboxylation to proceed and to attach the acetyl group to E1.

The mechanism of the acetyl transfer: the lipolysyl arm’s cyclic disulfide reduces to attach the acetyl group and get it away from E1, with a hemiacetyl-type intermediate.

The mechanism of the acetyl transfer: finally, the acetyl group is transferred to a more nucleophilic thiol on coenzyme A, generating acetyl-CoA.

To reset E2, E3 reduces the cyclic disulfide on E2 using the electron-carrying ability of the FAD it binds and a disulfide bridge; in turn, E3 oxidizes by reducing a NAD +.

In summary:

Active site of E3: the disulfide bridge at Cys 43 and 48, and a “separator” Tyr 181, which moves to allow the transfer of electrons.

Side note: arsenic is toxic because it bonds to the two sulfurs on molecules like lipoamides, and thus bring respiration to a halt More toxic to micro-organisms than humans, so arsenic- containing compounds were used as antibiotics and may have played a role in the deaths of Napoleon and Darwin.

Step 1: oxaloacetate + acetyl CoA citrate The enzyme is citrate synthetase.

Karpusas, M., Branchaud, B., and Remington, S.J., Proposed mechanism for the condensation reaction of citrate synthase: 1.9-.ANG. structure of the ternary complex with oxaloacetate and carboxymethyl coenzyme A, Biochemistry, 1990, 29 (9), pp. 2213–2219. Ordered Sequential mechanism: oxaloacetate binds first, then acetyl-CoA

Step 2: citrate isocitrate The enzyme is aconitase, which contains [4Fe-4S] iron sulfur clusters, which coordinate the OH on the citrate

Step 3: isocitrate α-ketoglutarate The enzyme is isocitrate dehydrogenase, which also requires NAD + and Mn 2+ (or Mg 2+ ); Tyr 160 and Lys 230 are conserved

The divalent cation stabilizes the newly-formed carbonyl

Step 4: α-ketoglutarate succinyl-CoA The enzyme is α-ketoglutarate dehydrogenase, which resembles and uses some subunits of the pyruvate dehydrogenase complex

Step 5: succinyl-CoA succinate The enzyme is succinyl-CoA synthetase = succinate thiokinase Three steps to transfer energy from the high- energy molecule succinyl-CoA to GTP; note the ability of the enzyme to bind phosphate

Step 6: succinate fumarate The enzyme is succinate dehydrogenase which has a FAD prosthetic group that accepts two electrons when the alkene is produced

Step 6: succinate fumarate The enzyme is succinate dehydrogenase which has a FAD prosthetic group that accepts two electrons when the alkene is produced The FAD is attached at a His residue

Step 7: fumarate malate The enzyme is fumarase = fumarate hydratase

Step 8: malate oxaloacetate The enzyme is malate dehydrogenase which requires NAD +

Energy and carbon summary of the citric acid cycle

Energy equivalents: 1 NADH = 2.5 ATP 1 FADH 2 = 1.5 ATP 1 GTP = 1 ATP These equivalences are strictly for energy and are approximate. In comparison to glycolysis, which produced the equivalent of 7 ATP, the citric acid cycle produces 25.

I’m guessing that at this point, you can figure out the irreversible (and therefore regulatory) steps in the citric acid cycle from this table.

Yup, these

Regulation of the pyruvate dehydrogenase complex 1.Inhibited by products NADH and acetyl-CoA 2.Covalent modification (phosphorylation and dephosphorylation) of E1

Regulation of the citric acid cycle Green indicates activators; red indicates steps that are inhibited by the substances connected by dashed lines.

Key point: the critical regulators of the citric acid cycle are its substrates (oxaloacetate and acetyl-CoA) and its product (NADH) For instance, to produce oxaloacetate (step 8), this is the equilibrium expression: If the cell runs low on NADH, then [oxaloacetate] increases to stimulate the cycle to produce more NADH.

Other pathways related to the citric acid cycle Cataplerotic reactions help remove the buildup of citric acid cycle intermediates: glucose biosynthesis, fatty acid biosynthesis, amino acid biosynthesis

Anaplerotic reactions build up citric acid cycle intermediates: pyruvate  oxaloacetate Pyruvate carboxylase transaminase

Glyoxalate cycle: plants, fungi and bacteria but not animals have the enzyme to aid conversion of acetyl-CoA to oxaloacetate Mediated in an organelle called the glyoxysome; two new enzymes (blue boxes)plus glyoxylate as an intermediate. The net effect is that two acetyl-CoA turn into a succinate.

How did the citric acid cycle evolve? Most anaerobes lack the citric acid cycle, but do have the enzymes that catalyze the last four reactions (highly conserved) where NAD + is reduced.