Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431


Fall 2008

Lecture Notes: 17 November

© R. Paselk 2008


Kreb's TCA Cycle

The Tricarboxylic acid cycle is in many ways the central pathway of metabolism, both catabolically and anabolically: it is involved in the breakdown and synthesis of a variety of compounds. Right now we want to focus on its catabolic role in aerobic catabolism: the oxidative breakdown of the acetyl group of acetyl CoA. In this instance we can consider the entire cycle to be a catalyst for this breakdown.

The problem is that the C-C bond of the acetyl group is chemically very resistant. Recall that in organic chemistry generally get C-C bond cleavages at alpha-beta bonds to carbonyl carbons, but with the acetyl group there is no beta carbon. So the TCA Cycle creates an alpha-beta bond by first attaching the acetyl group onto a carrier molecule, oxaloacetate.

Note that for pathways with a carier the carrier must be regenerated to consider the pathway completed!

Let's look at an overview of the Kreb's TCA Cycle. First condense the acetyl group with a four carbon carrier to get a six carbon tri-acid. This is then rearranged and oxidized with loss of carbon dioxide to give a five carbon di-acid ketol very similar to pyruvate in structure. An irreversible DH Complex then creates a four carbon CoA derivative with the release of a second carbon dioxide. At this point it appears that acetyl has been released as carbon dioxide, however, the carrier has been reduced, and modified. A series of reactions now regenerates the original carrier.

The first reaction of the cycle is an aldol condensation catalyzed by

Citrate synthase:

structural diagram of the catalytic mechanism of the attack of the coenzyme A carbanion on oxaloacetate in the enzyme Citrate synthase
Note that the enzyme catalyst enables the coupling of two chemically independent reactions: the aldol condensation (with free energy change of about zero) to the very favorable hydrolysis of the CoA thiol ester bond which drives the overall reaction far towards product. Essentially, we have used an ATP's worth of energy to drive the reaction to completion.


Unfortunately the resulting citrate is a tertiary alcohol which cannot be readily oxidized. Aconitase catalyzes the rearrangement of citrate to give an oxidizable secondary alcohol. This reaction involves an elimination/addition sequence, catalyzed by an iron-sulfur cluster (Fe4S4), with an alkene intermediate, cis- Aconitate:

structural diagram of the reaction catalyzed by the enzyme Aconitase including the aconitate intermediate

We have now converted the 3° alcohol, citrate, into an oxidizable 2° alcohol, isocitrate. The next reaction is the first oxidation of the TCA Cycle.

Isocitrate DH

The isocitrate alcohol can now be oxidized with NAD+ by Isocitrate DH to give an enzyme bound intermediate. The intermediate has a carboxyl group beta to a carbonyl carbon, so it has an excellent leaving group, CO2, attached to a stabilized carbanion. Thus it immediately rearranges to lose carbon dioxide:

structural diagram of the reaction catalyzed by the enzyme Isocitrate DH including the oxidized enzyme-bound intermediate

Alpha-Ketoglutarate DH Complex

The resulting 2-oxo-glutarate (-ketoglutarate) looks just like pyruvate with an R-group attached to the Beta-carbon, so it is broken down by a DH Complex, the Alpha-Ketoglutarate DH Complex, just as pyruvate was. As a DH Complex this is an irreversible enzyme, making the entire cycle irreversible. This gives succinyl-CoA and releases a second carbon dioxide. Note that at this point two carbons have been released, so formally, we have released the two carbons of Acetyl-CoA (Though neither of them came from the acetyl CoA we added)! We have also produced two NADH's (4 NADH/Glucose) which will result in the production of 5 ATP's (or: 4 x 2.5 ATP/NADH= 10 ATP's/Glucose). However, we have not regenerated the carrier. The remainder of the cycle is involved in this regeneration. (Again in the first four reactions two carbons have been lost as CO2 - as many carbons have been lost as were picked up with acetate. In a sense the rest of the cycle is regenerating our carrier - oxalacetate!)

Succinyl-CoA Synthetase

Succinyl-CoA, like acetyl-CoA, has a high-energy bond. However in this case the energy will be captured, using Succinyl-CoA Synthetase, to give a GTP which is energetically equivalent to an ATP (2 ATP's/Glucose). The mechanism of this reaction first involves the phosphorolysis by inorganic phosphate of the thiol ester bond to give a phosphoric-carboxylic mixed acid anhydride, followed by formation of a phosphorylated enzyme and finally transfer of the phosphate onto GDP.

Mainline Sequence reactions

The reactions beginning with succinate are representative of a common pattern, the "Mainline Sequence," seen repeatedly in biochemical pathways.

structural diagram of the "Mainline sequence" of reactions

Pathway Diagrams

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Last modified 18 November 2008