1. Ferchmin 2011 I ndex: 1.Pyruvate dehydrogenase complex, structure, cofactors, mechanism, regulation. 2.TCA or Kreb’s cycle, function and regulation. 3.Asymmetric breakdown of a symmetric citrate molecule. 4.“Accounting” of ATP synthesized, from glycolysis to respiratory chain. 5.Murphy’s Law and the missing 10 molecules of ATP per glucose. 6.How to avoid losing your carbons during the exam.

2. Steps shown and enzymes involved. 1 and 2) Pyruvate dehydrogenase (not decarboxylase). 3 and 4) Dihydrolipoyl transacetylase. 5) Dihydrolipoyl dehydrogenase Fully reduced lipoic How come NAD + +H transfers H to FAD? The –SH reduces the other –S- of lipoic acid

3. The pyruvate dehydrogenase complex is s true multienzymatic complex Electron micrography of the PDC of E. coli. Model of the PDC of E. coli. Red: E 1 pyruvate dehydrogenase Yellow: E 2 transacetylase Green: E 3 dihydrolipoyl dehydrogenase

4. 1 2 3 4 6 5

5. Regulation of the PDC by allosteric and covalent modulation The enzymes involved CoenzymesDescriptionActivatorsInhibitors 1) Pyruvate dehydrogenase (E 1 ) Thiamine pyrophosphate (Vitamin B1) Decarboxylation of pyruvate, E 1 α and E 1 β, phosphorylation site of PDC. 30 subunits AMP energy GTP energy 2) Lipoate transacetylase (E 2 ) Lipoic acidDehydrogenation of hydroxyethyl and transfer of acetyl to CoA. 60 subunits CoA TCA substrate CoA~Acetyl TCA substrate 3) Dihydrolipoyl dehydrogenase (E 3 ) NAD + and FAD (Niacin & B2) Regeneration of the lipoate and reduction of NAD + to NADH. 10 subunits NAD + Reducing power NADH; Reducing power The regulatory subunits of the PDC are intrinsic to the complex 1) Pyruvate dehydrogenase kinase inactivates the PDC Complex by phosphorylation of three serines on E 1 α. PRODUCTS ATP Acetyl CoA NADH SUBSTRATES ADP; pyruvate; CoA; NAD + 2) Pyruvate dehydrogenase phosphatase removes the inhibition imposed by the phosphorylation through hydrolysis of the P i. Ca 2+ insulin NADH Ca 2+ in muscle & insulin in liver Insulin (means abundance of glucose) disinhibits the PDC and reroutes pyruvate from gluconeogenesis to lipogenesis

6. Stoichiometry of the Krebs Cycle: CH3-CO-CoA + 3 NAD+ FAD + GDP + Pi + 2 H 2 O  2 CO 2 + 3 NADH + FADH 2 + GTP + 2 H + + CoA ΔG’°=1.1 kcal/mole (sluggish) but citrate leaves the mito. and serves as substrate for lipid synthesis Ac~CoA never leaves mito to favor TCA Succinyl~CoA accumulates in mitosol and serves as feedback inhibitor of citrate synthase, donor of CoA~ to activate ketone bodies and fatty acids and is also a precursor of porphyrines. Isocitrate dehydrogenase is the key enzyme (committed step). Strictly dependent on the ration of ADP/ATP and NAD + /NADH. Makes TCA aerobic by the “substrate control”. Odd carbon number fatty acids enter here and contribute to gluconeogenesis exiting the TCA as malate. Availability of oxaloacetate (OAA) is one of the main limiting steps of TCA. The [OAA] is 1/10 of the other intermediates of TCA. Remember pyruvate carboxylase is anaplerotic. Why Acetyl- CoA activates pyruvate carboxylase? Do you remember from glycolysis that the active metabolite (glyceraldehyde) is often kept in short supply? Succinate dehydrogenase is part of the mito. membrane and the respiratory chain Malate leaves the mitosol and goes into gluconeogenesis Very similar to PDC but has no intrinsic protein kinases & phosphatases. Otherwise has ~ the same regulation The green arrow indicates that the equilibrium is displaced towards malate.

7. Aconitase is inhibited by fluoroacetate an enzyme activated inhibitor (often called suicidal enzyme inhibitor). Malonic (HOCO-CH 2 -COOH) acid is the archetypal example of a competitive inhibitor: it acts against succinate dehydrogenase (complex II) in the respiratory electron transport chain. Odd carbon acids converts into propionyl- CoA which cannot directly enter either beta oxidation or the citric acid cycles. Instead it is carboxylated to D-methylmalonyl-CoA, which is isomerized to L-methylmalonyl- CoA. A vitamin B12-dependent enzyme catalyzes rearrangement of L- methylmalonyl-CoA to succinyl-CoA, which is an intermediate of the citric acid cycle.

8. This slide is for you to review the activators and inhibitors

9. The cycle is totally aerobic because of substrate (availability) control. Notice however, that O 2 is not actually present. The * indicate the requirement of an oxidized NAD + or FAD for the TCA to proceed. The II indicate the requirement of ADP for the cycle to proceed.

10. The puzzle During the first turn of the cycle no carbon from the Acetyl-CoA is released as CO2. Since citrate is symmetric this is impossible. Is citrate symmetric? What is going on?

11. Although citrate is symmetric it has two asymmetric mirror images. The concept is shown below. Only one side of citrate can bind cis-aconitase This is explained in the next slide

14. The yield of ATP per NADH+H + depends on the shuttle used.