Chapter 13 TCA Cycle The third fate of glucose/pyruvate is complete oxidation to C 2 + H 2 in the matrix of the mitochondrion. The 1 st step is the oxidation and decarboxylation of pyruvate to AcetylCoA, a form of activated acetate: CH 3 CC CoASH pyruvate deh2ase TPP, Lipoate, FAD NAD + ΔG ' o = 33.4 kj/mol K eq = 7x10 5 NADH + H + CH 3 C SCoA + C 2 Remember, there are 2 pyruvates from each glucose. In E. coli, pyruvate dehydrogenase is a large complex of 3 enzymes: 1
E 1 = pyruvatedeh 2 ase 24 copies E 2 = dihydrolipoyltransacetylase 24 copies E 3 = dihydrolipoyldeh 2 ase 12 copies It uses 5 coenzymes; 4 are derived from Vitamins: TPP à Thiamin = Vitamin B 1 FAD à Riboflavin = Vitamin B 2 NAD à Niacin = Vitamin B 3 CoA à Pantothenate = Vitamin B 5 Lipoate Coenzyme A Pantothenic Acid CH 3 βmercaptoethylamine C CHCNH C NH SH CH H 3 H 2 N P N N Adenine P N N P H Ribose 3' phosphate 2
Lipoic Acid CH S 2 S CH CH2 Lipoic Acid C HN CH2 Lys of E 2 CH N H C E 2 The reaction starts on E 1 and ends on E 3. The long flexible lipoic acid arm carries 2e from E 1 to E 3. E 1 uses TPP to decarboxylate pyruvate exactly as for pyruvate decarboxylase. Next, the lipoic acid on E 2 transfers the acetate from E 1 to CoA. Then, the lipoic acid is reoxidized by the FAD on E 3. Finally, the FADH 2 is reoxidized by NAD + and NADH carries the electrons away. 3
The reaction is irreversible and an important control point linking glycolysis and the TCA Cycle. It is inhibited by ATP, acetylcoa, NADH, fatty acids, C 2 highenergy signals It is activated by Pyruvate, AMP, CoA, NAD + lowenergy signals 4
In eukaryotes, the Citric Acid Cycle / Krebs Cycle / Tricarboxylic Acid Cycle acetate is oxidized to C 2 and H 2. The acetate may come from oxidation of glucose, amino acids, or lipids. The intermediates are used in AA, carbohydrate, pyrimidine nucleotide and lipid synthesis. Many of these enzymes are found in bacteria but bacteria rarely have the full cycle. 5
C C C Step 1: citrate synthase CH 3 C SCoA + H xaloacetate 2 ΔG 'o = 32.2 kj/mol K eq = 4x10 5 HSCoA + H + H C C C C Citrate [AA] is normally quite low so the G of thioester hydrolysis is used to drive the reaction forward. Citrate is a tricarboxylic acid. Citrate synthase is inhibited by ATP, NADH, AcetylCoA, SuccinylCoA and Citrate. 6
Step 2: aconitase H 2 H 2 ΔG 'o = +13.3 kj/mol K eq = 5x10 3 H H C C C C H C Isocitrate Aconitase catalyses 2 reactions that result in the isomerization of citrate to isocitrate: C 6 H 5 7 à C 6 H 5 7 The reaction is pulled forward by the following exergonic steps which consume isocitrate. isocitrate deh 2 ase Step 3: C H + + NAD + NADH + H + ΔG 'o = 20.9 kj/mol K eq = 5x10 3 C 2 C C αketoglutarate This reaction is an oxidation and a decarboxylation utilizing NAD + or NADP +. It is inhibited by ATP and activated by ADP. 7
Step 4: αketoglutarate deh 2 ase complex TPP, Lipoate, FAD CoASH NAD + C 2 NADH ΔG 'o = 33.5 kj/mol K eq = 7x10 5 C C S CoA SuccinylCoA The mechanism is identical to the pyruvate dehydrogenase reaction. This reaction is an oxidation and a decarboxylation. Some of the G of oxidation is conserved in the formation of a thioester bond of succinylcoa. This enzyme is inhibited by NADH and succinylcoa. 8
Step 5: succinylcoa synthetase C GDP + P i GTP ΔG 'o = 2.9 kj/mol K eq = 3 CoASH C Succinate The G released when the highenergy thioester is hydrolysed is conserved in the formation of GTP. Substrate Level Phosphorylation Remember, GTP and ATP are energetically equivalent. Step 6: succinate deh 2 ase FAD FADH 2 ΔG 'o = 0 kj/mol K eq = 1 C CH CH C Fumarate The G of oxidation is stored in reduced FAD which is covalently attached to the enzyme. 9
fumarase H 2 ΔG 'o = 3.8 kj/mol K eq = 5 Step 7: C H CH H C H C LMalate Fumarase stereospecifically adds water across the C=C bond. malate deh 2 ase Step 8: C H C H NAD + NADH + H + C C ΔG 'o = +29.7 kj/mol K eq = 6x10 6 xaloacetate This oxidation is highly endergonic so [AA] is always low. Exergonic reaction 1 pulls this forward. About 50 kj /mol of G is released by the cycle and drives it in the direction of products. 10
Energy and Mass Balance AcetylCoA + 2H 2 + 3NAD + + FAD + GDP + P i à 2C 2 + CoASH + 3NADH + 2H + + FADH 2 + GTP Input utput 1 acetate = 2 C + 1 2 C 2 i.e. 2 C and 4 ; 2 H 2 = 2 from AA, not from the P i = 1 1 acetate added at the beginning of the cycle. 4 steps involve oxidations that conserve G by reducing electron carriers (3 NADH + 1 FADH 2 ) plus 1 high energy phosphate is formed (GTP). Note that reactions 68 regenerate AA so there is no net consumption or production of intermediates. The cycle functions as a catalyst. Why is 2 required? The cycle would stop if NAD + were not regenerated: NADH + H + + ½ 2 à H 2 + NAD + The transport of electrons from NADH to 2 is coupled to ATP formation. 11
ADP + P i + H + ATP + H 2 The process is called oxidative phosphorylation and is the subject of Chapter 14. 12