Thiamine pyrophosphate and lipoic acid

This looks like a simple reaction based on very small molecules. But look again. It Is a very strange reaction indeed. The molecule of CO2 clearly comes from the carboxyl group of pyruvate, but how is the C-C bond cleaved, and how does acetyl CoA Join on If you try to draw a mechanism you will see that there must be more to this reaction than meets the eye. The extra features are two new cofactors, thiamine pyrophosphate and lipoic acid, and the reaction takes place in several stages with some interesting chemistry involved. 

The metabolic functions of pantothenic acid in human biochemistry are mediated through the synthesis of CoA. Pantothenic acid is a structural component of CoA. which is necessary for many important metabolic processes. Pantothenic acid is incorporated into CoA by a. series of five enzyme-catalyzed reactions. CoA is involved in the activation of fatty acids before oxidation, which requires ATP to form the respective fatty ocyl-CoA derivatives. Pantothenic acid aI.so participates in fatty acid oxidation in the final step, forming acetyl-CoA. Acetyl-CoA is also formed from pyruvate decarboxylation, in which CoA participates with thiamine pyrophosphate and lipoic acid, two other important coenzymes. Thiamine pyrophosphate is the actual decarboxylating coenzyme that functions with lipoic acid to form acetyidihydrolipoic acid from pyruvate decarboxylation. CoA then accepts the acetyl group from acetyidihydrolipoic acid to form acetyl-CoA. Acetyl-CoA is an acetyl donor in many processes and is the precursor in important biosyntheses (e.g.. those of fatty acids, steroids, porphyrins, and acetylcholine). 

As is so often the case with multiple-enzyme systems, in beginning studies investigators needed a particulate enzyme preparation to demonstrate the reaction. Later, however, a soluble enzyme preparation became available, and it was demonstrated that the various steps of the reaction are performed by a high molecular weight compound to which thiamine pyrophosphate and lipoic acid are attached. The detailed mechanism of the reaction is far from understood it is not even known how many enzymes are involved. In bacteria, the complex has been resolved into two separate fractions named A and B, and neither fraction was found to be active by itself. In other words, a-ketoglutarate is oxidized and decarboxylated only if the A and B fractions are recombined. 

Figure 7-1. Conversion of pyruvate to acetyl CoA by the pyruvate dehydrogenase complex. The three enzymes, pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase, exist in a complex associated with the mitochondrial matrix. Each enzyme requires at least one coenzyme that participates in the reaction. TPP, thiamine pyrophosphate Lip, lipoic acid CoA, coenzyme A.

Pyruvate produced by the glycolytic pathway may be transported into the mitochondria (via an antiport with OH"), where it is converted to acetyl-CoA by the action of the enzyme complex pyruvate dehydrogenase. The pertinent enzyme activities are pyruvate dehydrogenase (PD), lipoic acid acetyltransferase, and dihydrolipoic acid dehydrogenase. In addition, several cofactors are utilized thiamine pyrophosphate (TPP), lipoic acid, NAD+, Co A, and FAD. Only Co A and NAD+ are used in stoichiometric amounts, whereas the others are required in catalytic amounts. Arsenite and Hg2+ are inhibitors of this system. The overall reaction sequence may be represented by Figure 18.5. The NADH generated may enter the oxidative phosphorylation pathway to generate three ATP molecules per NADH molecule reduced. The reaction is practically irreversible its AGq = -9.4 kcal/mol. 

The mechanism of the pyruvate dehydrogenase reaction is wonderfully complex, more so than is suggested by its simple stoichiometry. The reaction requires the participation of the three enzymes of the pyruvate dehydrogenase complex and five coenzymes. The coenzymes thiamine pyrophosphate (TPP), lipoic acid, and FAD serve as catalytic cofactors, and CoA and NAD" are stoichiometric cofactors. 

There are two 2-oxoacid dehydrogenase multienzyme complexes in E. coli. One is specific for pyruvate, the other for 2-oxoglutarate. Each complex is about the size of a ribosome, about 300 A across. The pyruvate dehydrogenase is composed of three types of polypeptide chains El, the pyruvate decarboxylase (an a2 dimer of Mr — 2 X 100 000) E2, lipoate acetyltransferase (Mr = 80 000) and E3, lipoamide dehydrogenase (an a2 dimer of Mr = 2 X 56 000). These catalyze the oxidative decarboxylation of pyruvate via reactions 1.6, 1.7, and 1.8. (The relevant chemistry of the reactions of thiamine pyrophosphate [TPP], hydroxyethylthiamine pyrophosphate [HETPPJ, and lipoic acid [lip-S2] is discussed in detail in Chapter 2, section C3.)... 

However, this reaction is in fact the sum of five reactions catalyzed by three enzymes and requiring five cofactors coenzyme A (CoA), NAD+, FAD, thiamine pyrophosphate (TPP), and lipoic acid (an 8-carbon acid with sulfhydryl groups at positions 6 and 8). The detailed steps of these reactions are shown in Fig. 12-7. 

At the centre is enzyme 2 which binds the acetyl group through a lipoic acid-lysine amide. On the one side this acetyl group is delivered from pyruvate by the ministrations of thiamine pyrophosphate and enzyme T and on the other it is delivered to CoA as the free thiol ester. Enzyme 3 recycles... 

See also Thiamine Pyrophosphate and Decarboxylations, Lipoic Acid, Ethanol Fermentation, Pyruvate Dehydrogenase, Pyruvate Decarboxylase, Acetaldehyde, ot Ketoglutarate Dehydrogenase... 

We have already mentioned two vitamin-derived cofactors involved in the PDH reaction (CoA and NAD+), but in addition three others are involved - thiamine pyrophosphate (TPP) from vitamin B1, FAD from riboflavin (vitamin B2) and lipoic acid, so the PDH step depends on flve different vitamins and would fail if any of them were missing. 

The oxidative decarboxylation of pyruvate is considerably more complex than is suggested by the preceding equation. In addition to utilizing NAD and coenzyme A, this transformation also requires FAD, thiamine pyrophosphate (which is derived from thiamine, vitamin Bl), and lipoic acid ... 

The reactions in which pyruvic acid is oxidized to form CO2 and acetic acid are of the greatest significance since they constitute the link between glycolysis and the Krebs cycle. These reactions involve a considerable number of coenzymes thiamine pyrophosphate, lipoic acid, Co A, and NAD. Much of our knowledge of pyruvic acid oxidation depended on the discovery of CoA and lipoic acid, and it might be useful to review the biochemistry of lipoic acid before we enter into more detail. Refer to the chapter on vitamins for a review of the metabolism and catabolism of thiamine, CoA, and NAD. 

Several metabolic blocks could account for the biochemical distortion observed in maple syrup disease. A deficiency in amino oxidase could lead to accumulation of amino acids. Because the enzyme has such a broad specificity, whenever it is completely deleted a more complex aminoaciduria can be expected to develop. The deletion of a specific transaminase could hardly explain the keto acid accumulation. Therefore, it seems more likely that the metabolic block involves a step between the keto acid and the simple acids, possibly the oxidative decarboxylation of the keto acid. This reaction requires coenzyme A, NAD, lipoic acid, and thiamine pyrophosphate, and it was described in some detail in the chapter devoted to the bioenergetic pathways. Leukocytes of at least some patients with maple syrup disease have been shown to contain normal transaminase activity but are defective in the oxidative decarboxylase. 

Fatty acids are synthesized in a multienzyme complex from a crucially important primary metabolite, acetyl-coenzyme A (7.5). The principal source of acetyl-CoA (7.5) is pyruvic acid (7.5) and the conversion of (7.5) into (7.5) involves the coenzymes, thiamine pyrophosphate (7.5) and lipoic acid (7.5) (Scheme 1.1). The key to the action of thiamine is the ready formation of the zwitterion 1.4) at the beginning and end of the reaction cycle. The lipoic acid (7.5) is enzyme linked via the side chain of a lysine residue (7.7). The disulphide functionality is thus at the end of a long (14 A) arm. It has been suggested that this arm allows the lipoate to swing from one... 

The pyruvate dehydrogenase complex (PDC) is a noncovalent assembly of three different enzymes operating in concert to catalyze successive steps in the conversion of pyruvate to acetyl-CoA. The active sites of ail three enzymes are not far removed from one another, and the product of the first enzyme is passed directly to the second enzyme and so on, without diffusion of substrates and products through the solution. The overall reaction (see A Deeper Look Reaction Mechanism of the Pyruvate Dehydrogenase Complex ) involves a total of five coenzymes thiamine pyrophosphate, coenzyme A, lipoic acid, NAD+, and FAD. 

The mechanism of the pyruvate dehydrogenase reaction is a tour de force of mechanistic chemistry, involving as it does a total of three enzymes (a) and five different coenzymes—thiamine pyrophosphate, lipoic acid, coenzyme A, FAD, and NAD (b). 

The first step of this reaction, decarboxylation of pyruvate and transfer of the acetyl group to lipoic acid, depends on accumulation of negative charge on the carbonyl carbon of pyruvate. This is facilitated by the quaternary nitrogen on the thiazolium group of thiamine pyrophosphate. As shown in (c), this cationic... 

Group-transfer reactions often involve vitamins3, which humans need to have in then-diet, since we are incapable of realizing their synthesis. These include nicotinamide (derived from the vitamin nicotinic acid) and riboflavin (vitamin B2) derivatives, required for electron transfer reactions, biotin for the transfer of C02, pantothenate for acyl group transfer, thiamine (vitamin as thiamine pyrophosphate) for transfer of aldehyde groups and folic acid (as tetrahydrofolate) for exchange of one-carbon fragments. Lipoic acid (not a vitamin) is both an acyl and an electron carrier. In addition, vitamins such as pyridoxine (vitamin B6, as pyridoxal phosphate), vitamin B12 and vitamin C (ascorbic acid) participate as cofactors in an important number of metabolic reactions. 

Fig. 9. A schematic drawing of a possible mechanism for the reaction catalyzed by the pyruvate dehydrogenase complex. The three enzymes Elf E2, and E3 are located so that lipoic acid covalently linked to E2 can rotate between the active sites containing thiamine pyrophosphate (TPP) and pyruvate (Pyr) on Elt CoA on E2, and FAD on E3. Acetyl-CoA and GTP are allosteric effectors of E, and NAD+ is an inhibitor of the overall reaction.

Coenzymes The pyruvate dehydrogenase complex contains five coenzymes that act as carriers or oxidants for the intermediates of the reactions shown in Figure 9.3. Ei requires thiamine pyrophosphate, Ep requires lipoic acid and coenzyme A, and E3 requires FAD and NAD+. [Note Deficiencies of thiamine or niacin can cause serious central nervous system problems. This is because brain cells are unable to produce sufficient ATP (via the TCA cycle) for proper function if pyruvate dehydrogenase is inactive.]... 

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