Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431	Biochemistry	Fall 2001
Lecture Notes:: 12 November	© R. Paselk 2001
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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 of 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 a-b bonds to carbonyl carbons, but with acetyl group there is no beta carbon. So the TCA Cycle creates an a-b bond by first attaching the acetyl group onto a carrier molecule, oxaloacetate.

 

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. A series of reactions then regenerates the original carrier.

 

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

 

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. Unfortunately the resulting citrate is a tertiary alcohol which cannot be readily oxidized, so the next enzyme,

 

Aconitase, catalyzes its rearrangement 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:

 

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.

The isocitrate alcohol can now be oxidized by Isocitrate DH to give an enzyme bound intermediate, with a carboxyl group b to a carbonyl carbon, which immediately rearranges to lose carbon dioxide:

The resulting 2-oxo-glutarate (a-ketoglutarate) looks just like pyruvate with an R-group attached to the b-carbon, so it is broken down by a DH Complex, a-Ketoglutarate DH Complex, just as pyruvate was. 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.

Note 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, like acetyl-CoA, has a high-energy bond. However in this case the energy will be captured to give a GTP which is energetically equivalent to an ATP (2 ATP's/Glucose). The mechanism of this reaction, which 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.

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

First, Succinate DH, an inner-mitochondrial membrane-bound enzyme and member of the mitochondrial electron transport system (ETS), oxidizes succinate to fumerate. This reaction uses the stronger oxidizer FAD as an oxidizing agent because of the added difficulty in oxidizing an alkane to an alkene. As a consequence of using this more powerful oxidizing agent, less ATP energy can be captured in oxidizing the resulting FADH₂ with oxygen (FAD is closer to oxygen in its oxidation potential). One and one-half ATP equivalents are obtained in this reaction (or: 2 x 1.5 ATP/FADH₂= 3 ATP/Glucose).

The resulting alkene, Fumerate, is not readily oxidized. However, if water is added across the double bond an alcohol results which can be oxidized. Thus Fumerase catalyses a hydration reaction to give malate.

Finally, Malate DH catalyzes the dehydrogenation of malate to regenerate the original carrier, oxaloacetate, and finish the cycle. In addition another NADH is formed (and 2 x 2.5 ATP/NADH= 5 ATP/Glucose).

For the entire cycle we then have the production of 10 ATP/acetyl-CoA or 20 ATP/Glucose. The aerobic catabolism of glucose can then give a maximum total of 32 ATP/glucose as summarized in the Table:

* In some tissues (insect flight muscle, fast twitch muscle) the reducing equivalents of NADH must be pumped against a gradient at a cost of 1 ATP (it is used to make FADH2).  Reaction Energy Product factor ATP Equivalents (@2.5 ATP/NAD) ATP Equivalents (@3 ATP/NAD)  Hexokinase ADP 1 x -1 - 1 -1   PFK ADP 1 x-1 - 1  -1  GA-3-P DH NADH 2 x 2.5 (1.5)* 5 (3)* 6 (4)* PGA Kinase ATP 2 x1 2  2 Pyruvate Kinase ATP 2 x 1 2  2 Pyruvate DH Complex NADH 2 x 2.5 5  6 Isocitrate DH NADH 2 x 2.5 5 6  2-oxoglutarate DH Complex NADH 2 x 2.5 5  6 Succinyl-CoA Synthetase GTP 2 x 1 2 2  Succinate DH FADH2 2 x 1.5 3  4 Malate DH NADH 2 x 2.5 5  6   TOTAL= 32 (30)*  38 (36)*

Pathway Diagrams

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Last modified 12 November 2001