Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438 - Introductory Biochemistry - Spring 2013

Lecture Notes:

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Pyruvate Metabolism

Let's turn now to the fate of pyruvate in aerobic tissues. Pyruvate must first be transported into the mitochondria, where it can then be oxidized to give acetyl CoA, which can then be used to make fat for storage or it can be further oxidized to carbon dioxide via the Kreb's TCA Cycle.

FYI - DH Complex Protein Structure

Structure of Pyruvate DH Complex from bovine kidney: MW = 7 x 106 (without associated Phosphatase)

  1. Transacetylase: 60 subs x 52,000 = 3.1 x 106 arranged as a pentagonal dodecahedron
  2. Dihydrolipoyl DH: 5 dimers x 110,000 = 5 x 105 on faces of dodecahedron
  3. Pyruvate DH: 10 tetramers x 154,000 = 1.54 x 106 on edges of dodecahedron

The oxidation of pyruvate to acetyl CoA is accomplished by the Pyruvate Dehydrogenase complex, a large, multi-component enzyme with three main enzyme subunits. The reactions of the Pyruvate DH Complex are outlined in the diagram.

The first enzyme of this complex, pyruvate dehydrogenase (note that, unusual for the DH appellation, there is no direct NAD+ or FAD involvement), catalyzes two sequential reactions. In the first reaction, catalyzed by the alpha subunit of the enzyme, the coenzyme Thiamine Pyrophosphate (TPP), with a highly acidic carbon (a stable carbanion), attacks pyruvate at C-2 with the loss of carbon dioxide to give a covalent coenzyme-substrate intermediate. In the second reaction, catalyzed by the beta subunit, the ketol group is oxidatively transferred to one of the sulfurs of the lipoyl coenzyme on the second enzyme of the complex, dihydrolipoyl transacetylase, to give an acetyl-lipoamide intermediate.

The lipoamide of dihydrolipoyl transacetylase constitutes a long arm which may now move the acetyl group from the active site of pyruvate DH to its own active site where the lipoamide is exchanged for Coenzyme A-SH. (On the mammalian enzyme the 60 subunits of the transacetylase seem to form a pool of lipoyl groups among which the acetyl groups are freely exchanged.)

structure of lipoate

Note that in the reactions of dihydrolipoyl transacetylase the lipoamide has been reduced from a disulfide to two sulfhydryl groups. In order to continue operation lipoamide must be reoxidized and that is accomplished by the final enzyme of the complex, dihydrolipoyl dehydrogenase. The reactions catalyzed by this enzyme are complex, but the net result is the transfer of two electrons from the lipoamide to NAD+ to give NADH.

Overall then the Pyruvate DH Complex converts pyruvate into acetyl CoA in a physiologically irreversible reaction with the release of carbon dioxide and the capture of an electron pair as a hydride ion on NADH. Note the cofactors involved for this reaction sequence: TPP, FAD, Mg2+, lipoamide, Coenzyme A, and NAD+

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-betabond 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. (Keep in mind that in looking at the Kreb's Cycle as a catalyst, all catalysts MUST be regenerated, that is we must return to our starting point in terms of shoichiometry. ) 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 for citrate synthase reaction

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.

Aconitase

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:

Aconitase reaction structures

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:

Isocitrate DH reaction including structures

Ketoglutarate DH Complex

The resulting 2-oxo-glutarate (alpha-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. 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.

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

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

generalized diagram for mainline sequence reactions

Succinate DH

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).

Fumerase

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.

Malate DH

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).

Energy Capture with the TCA Cycle

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)
Glycolysis
 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 & Kreb's Cycle
Pyruvate DH Complex
NADH

2 x 2.5

5
 6
Isocitrate DH
NADH

2 x 2.5

5
2-oxoglutarate DH Complex
NADH

2 x 2.5

5
 6
Succinyl-CoA Synthetase
GTP

2 x 1

2
Succinate DH
FADH2

2 x 1.5

3
 4
Malate DH
NADH

2 x 2.5

5
 6

  TOTAL=

32 (30)*
 38 (36)*

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© R. A. Paselk 2010;

Last modified 29 March 2013