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Bryan Krantz: University of California, Berkeley MCB 102, Spring 2008, Metabolism Lecture 8 Reading: Ch. 16 of Principles of Biochemistry , “The Citric Acid Cycle.”
CITRIC ACID CYCLE
Three Phases
[1] Acetyl-CoA production— Organic fuels (glucose, amino acids, fats) Acetyl-CoA [2] Acetyl-CoA oxidation— Acetyl-CoA enters TCA and is enzymatically oxidized; energy is conserved in electron carriers, NADH FADH 2 [3] Electron transfer— energy rich e-^ from NADH FADH 2 reduce O 2 to H 2 O
Pyruvate Dehydrogenase Complex
The first reaction before you enter the TCA cycle is the conversion of pyruvate into the two carbon intermediate that is necessary for entry into the cycle, acetyl-CoA – an acetate attached to Coenzyme A (CoA).
Pyruvate + NAD+^ + CoA Acetyl-CoA + NADH + CO 2
The enzyme is called Pyruvate Dehydrogenase Complex , which contains many coenzymes and co-factors. The simplest pyruvate dehydrogenase enzyme in E. coli contains 60 subunits! Subunits possess 1 of 3 activities: E1, E2 and E3. Other enzyme complexes are related & similar.
Overall Energetics. Δ G º’ is – 33 kJ/mol—a strongly downhill reaction.
E
Pyruvate dehydrogenase is the actual dehydrogenase activity and first enzyme in the complex.
Pyruvate + TPP Hydroxyethyl TPP + CO 2
Mechanism. E1 uses coenzyme, thiamine pyrophosphate, or TPP, (from vitamin B1)—which is powerful electron sink. Mechanism is similar to aldolase, where electron withdrawing power aids in breaking the C-C bond.
Carbanion carbon of TPP attaches to the carbonyl carbon of the pyruvate. The purpose of doing that is to use the strong electron sink, which is the quaternary nitrogen, of thiazol to allow the decarboxylation of CO 2 , because CO 2 gets released. After decarboxylation and the addition of the proton, you get hydroxy-ethyl TPP. This reaction is like that for the decarboxylation of pyruvate to acetaldehyde.
Coenzyme A (CoA)
E
Dihydrolipoyl dehydrogenase enzyme oxidizes dihydrolipoyllysine by transferring the energy rich electrons to an electron carrier, NAD+, via a tightly bound intermediary electron carrier, FAD.
Dihydroxylipoyllysine + NAD+^ Lipoyllysine + NADH
FAD. The flavin group is the business end of FAD; it is not linked to ribose, but to ribitol—a reduced product of ribose. Then, it is linked to a pyrophosphate moeity, ribose and adenine. FAD comes from vitamin B 2. A closely related coenzyme called FMN constitutes one half of this molecule, where you cut after the first phosphate group.
First the electrons in the lipoyllysine are transferred to FAD. In the second reaction, the oxidized form of FADH 2 is regenerated. The reason for all this is that unlike NAD+/NADH that exist freely in solution, FAD and FMN usually exist in close association with enzymes, so they cannot float away. FAD is still connected tightly with the E enzyme.
[STEP 1] Citrate Synthase. The first reaction is a synthase reaction, called such since a new molecule is made but ATP is not used. (The latter is called a synthetase ).
Oxaloacetate + Acetyl-CoA + H 2 O Citrate + CoA
Mechanism. Claisen condensation reaction from organic chemistry, where thioester is combined with ketone. Methyl carbon of acetyl- CoA attack the electron poor ketone carbon of oxaloacetate. You have to abstract a proton, so you produce a carbanion here. The carbanion will attack this oxaloacetate carbonyl carbon. To produce carbanion, the enzyme uses the general acid/base mechanism with two His residues.
Energetics. The synthesis of the C-C bond here is probably should not be a strongly downhill reaction. To make this reaction strongly downhill, you use the high free energy difference gained by the hydrolysis of this thioester to drive the reaction. The thioester hydrolysis has a Δ G º’ = – 30 kJ/mol so exothermic energy difference is almost entirely coming from the hydrolysis of the thioester.
Historic fight. It was thought that the synthase step was not the entry step into the TCA Cycle. Citrate is a symmetric molecule (lacks a stereocenter.) However, radiolabeling studies showed that the two symmetric carboxyl groups were not treated the same. Later, this was resolved: citrate is close to being chiral ( prochiral ) and a prochiral substrate is treated as chiral if the enzyme is chiral.
[STEP 2] Aconitase.
Citrate [ H 2 O + cis- Aconitate ] Isocitrate
Mechanism. This is a dehydration reaction followed by a hydration. The dehydration step is like enolase in glycolysis. To facilitate the abstraction of the proton, you pull the electrons strongly from the carboxylate. Enolase did this by putting magnesium near the carboxylate oxygen. Aconitase uses an iron-sulfur cluster cofactor instead of magnesium to do this. Three Cys residues and multiple Fe atoms make the cluster.
Energetics. The equilibrium favors citrate ( ΔGº’ = 13 kJ/mol) but under normal physiological conditions, isocitrate is favored. How?
Other Regulation. Aconitases also regulate iron uptake and metabolism in the cell.
[STEP 4] α-Keto Glutarate Dehydrogenase. This enzyme splits the carbon-carbon bond and is related to pyruvate dehydrogenase.E1 and E2 are similar, and E3 is identical in sequence!
α-keto glutarate + CoA Succinyl-CoA
Mechanism. α-keto glutarate dehydrogenase works exactly like pyruvate dehydrogenase. You have the five coenzymes: TPP, lipoyllysine, CoA, FAD and NAD+. These are all used, and you get oxidation. The decarboxylated product occurs as a thioester. The product is succinyl-CoA. The thioester in the succinyl-CoA will be utilized later of course in an analogous manner.
[STEP 5] Succinyl-CoA Synthetase.
Succinyl-CoA + GDP + Pi Succinate + GTP
Mechanism. Phosphorylysis reaction is followed by phosphoryl transfer to GDP, producing succinate plus GTP. Note the phosphoryl group is transferred to the GDP via an intermediate that forms with a His residue on the enzyme’s active site.
Energetics. Capitalized on the CoA thioester by capturing free energy as a GTP. GTP is as good as ATP, because there is a free conversion of nucleoside triphosphates in the cell (by the enzyme, nucleoside diphosphate kinase ).
GTP + ADP GDP + ATP where ΔGº’ = 0 kJ/mol
[STEP 7] Fumarase. The next reaction is called fumarase , which converts fumarate into a hydroxy- dicarboxylic acid called malate. This is an addition of water across the double bond—a recurring theme in the course.
[STEP 8] Malate Dehydrogenase. Finally, there is malate dehydrogenase, which uses NAD+^ to oxidize this product malate to oxaloacetate. This process yields NADH.
● We talked about this reaction in gluconeogenesis as a way to get reducing equivalents into the cytosol. ● Here the reaction completes the TCA Cycle, remaking oxaloacetate.
