Thiamine coenzyme action

Structures of Thiamin-Dependent Enzymes 4. The Variety of Enzymatic Reactions Involving Thiamin 5. Oxidative Decarboxylation and 2-Acetylthiamin Diphosphate. 6. Thiamin Coenzymes in Nerve Action 753. .. Table 14-4 Some Pyruvoyl Enzymes... 

Coenzymes complement the catalytic action of the amino-acid functional groups. They are bound to apoenzymes (apoproteins) either covalently or non-covalently. It is interesting to note that non-covalently-bound coenzymes are polyanions at neutral pH as exemplified by the structures of glutathione (GSH) [17] and thiamine pyrophosphate [18]. Shinkai and Kunitake (1976b, 1977a) demonstrated the efficient binding of glutathione and coenzyme A (a polyphosphate) to cationic micelles and cationic polysoaps. Thus, the combina- ... 

The next coenzyme for which a mechanism was established was thiamin pyrophosphate [3]. Ronald Breslow used nmr spectroscopy to show that the hydrogen atom at C-2 of a thiazolium salt rapidly exchanges with deuterium in even slightly alkaline solutions (6), so that the coenzyme offers an anionic centre for catalysis (Breslow, 1957). With this established, Breslow could confidently offer the pathway shown in Scheme 2 for the action of the... 

In the first step, pyruvate is decarboxylated in an irreversible reaction catalyzed by pyruvate decarboxylase. This reaction is a simple decarboxylation and does not involve the net oxidation of pyruvate. Pyruvate decarboxylase requires Mg24" and has a tightly bound coenzyme, thiamine pyrophosphate, discussed below. In the second step, acetaldehyde is reduced to ethanol through the action of alcohol dehydrogenase, with... 

The combined dehydrogenation and decarboxylation of pyruvate to the acetyl group of acetyl-CoA (Fig. 16-2) requires the sequential action of three different enzymes and five different coenzymes or prosthetic groups—thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), coenzyme A (CoA, sometimes denoted CoA-SH, to emphasize the role of the —SH group), nicotinamide adenine dinucleotide (NAD), and lipoate. Four different vitamins required in human nutrition are vital components of this system thiamine (in TPP), riboflavin (in FAD), niacin (in NAD), and pantothenate (in CoA). We have already described the roles of FAD and NAD as electron carriers (Chapter 13), and we have encountered TPP as the coenzyme of pyruvate decarboxylase (see Fig. 14-13). 

We see that the essence of the action of thiamin diphosphate as a coenzyme is to convert the substrate into a form in which electron flow can occur from the bond to be broken into the structure of the coenzyme. Because of this alteration in structure, a bond breaking reaction that would not otherwise have been possible occurs readily. To complete the catalytic cycle, the electron flow has to be reversed again. The thiamin-bound cleavage product (an enamine) from either of the adducts in Eq. 14-20 can be reconverted to the thiazolium dipolar ion and an aldehyde as shown in step b of Eq. 14-21 for decarboxylation of pyruvate to acetaldehyde. 

Another early success in biomimetic chemistry concerns reactions promoted by thiamin. In 1943, more than 35 years ago, Ukai, Tanaka, and Dokowa (12) reported that thiamin will catalyze a benzoin-type condensation of acetaldehyde to yield acetoin. This reaction parallels a similar enzymic reaction where pyruvate is decarboxylated to yield acetoin and acetolactic acid. Although the yields of the nonenzymic process are low, it is clearly a biomimetic process further investigation by Breslow, stimulated by the early discovery of Ugai et al., led to an understanding of the mechanism of action of thiamin as a coenzyme. 

The coenzyme thiamine pyrophosphate (1) plays a central role in many parts of metabolism (Fig. 1). Its mechanism of action involves the formation of a thiazolium zwitterion 2 that was stabilized by a carbene resonance form 3 (10). This discovery opened up studies of the chemistry that such stabilized carbenes could catalyze, as chemists realized that the otherwise impossible chemistry that thiamine pyrophosphate catalyzes in nature could be generalized and adapted for useful synthetic processes. 

The C-2-exchange of azolium salts via an ylide mechanism was discussed in Section 24.1.2.1. Thiamin pyrophosphate acts as a coenzyme in several biochemical processes and in these, its mode of action depends on the intermediacy of a 2-deprotonated species (32.2.4). In the laboratory, thiazolium salts (3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium chloride is commercially available) will act as catalysts for the benzoin condensation, and in contrast to cyanide, the classical catalyst, allow such reactions to proceed with alkanals, as opposed to araldehydes the key steps in thiazolium ion catalysis for the synthesis of 2-hydroxy-ketones are shown below and depend on the formation and nucleophilic reactivity of the C-2-ylide. Such catalysis provides acyl-anion equivalents. 

The enyzyme uses thiamine pyrophosphate as a coenzyme (Figure 14.14) and is very similar in mechanism of action to the pyruvate dehydrogenase complex (Figure 14.10 ... 

Pyruvate decarboxylase uses thiamine pyrophosphate as a coenzyme and catalyzes the decarboxylation of pyruvate. This decarboxylated product has two fates. In yeast, acetaldehyde is formed, which is subsequently converted to ethanol by action of the enzyme alcohol dehydrogenase. In non-fermentative reactions, acetyl-CoA is formed. 

Although thiamine, a thiazolium salt, contains a pyrimine ring, it is the thiazole ring that is responsible for its biological action, thiamine dihosphate being the coenzyme of decarboxylases. The mechanism of the catalytic decarboxylation (e.g. of pyruvic acid to acetaldehyde) was interpreted by Breslow in 1958. The active species is the N-ylide 12 formed from thiamine diphosphate and basic cell components ... 

The first step in the reaction sequence that converts pyruvate to carbon dioxide and acetyl-CoA is catalyzed by pyruvate dehydrogenase, as shown in Figure 19.4. This enzyme requires thiamine pyrophosphate (TPP a metabolite of vitamin Bj, or thiamine) as a coenzyme. The coenzyme is not covalently bonded to the enzyme they are held together by noncovalent interactions. Mg + is also required. We saw the action of TPP as a coenzyme in the conversion of pyruvate to acetaldehyde, catalyzed by pyruvate decarboxylase (Section 17.4). In the pyruvate dehydrogenase reaction, an a-keto acid, pyruvate, loses carbon dioxide the remaining two-carbon unit becomes covalently bonded to TPP. 

Sulfur is a constituent of the amino acids, methionine, cystine and cysteine, which are essential in proteins supplied in animal diets. Sulfur is also a constituent of the vitamins, thiamine and biotin, and of glutathione and coenzyme A. It plays an important role in the action of proteolytic enzymes and in oxidation—reduction processes. As a constituent of proteins it is present in cell protoplasm and is therefore of vital importance in cell metabolic processes. 

Enzymes, also, are often so specific that the addition or elimination of a single methyl-group in a substrate or coenzyme can cause a large, or even complete, loss of reactivity. How the biological action of thiamine is affected by the addition or loss of a single methyl-group was described at the beginning of this Chapter. 

The C-2-exchange of azolium salts via an ylid mechanism has already been discussed (section 21.1.2.1). Thiamin pyrophosphate acts as a coenzyme in several biochemical processes and in these its mode of action also depends on the intermediacy of a 2-deprotonated species. For example, in the later stages of alcoholic fermentation, which converts glucose into ethanol and carbon dioxide, the enzyme pyruvate decarboxylase converts pyruvate into ethanal and carbon dioxide, the former then being converted into ethanol by the enzyme alcohol dehydrogenase. It is believed that, in the operation of the former enzyme, the coenzyme, thiamin pyrophosphate, adds as its ylid to the ketonic carbonyl group of pyruvate this is followed by loss of carbon dioxide, then the release of ethanal by expulsion of the original ylid. 

To elucidate the molecular mechanism of action of coenzymes, biochemists often use chemical models. The first such model in the case of thiamine was based on the observation that heat decarboxylates pyruvic acid, and that amines catalyze this reaction. However, thiamine is not the most effective catalyst in this type of reaction. 

Mizuhara [65] developed a more adequate model by demonstrating that thiamine catalyzes the decarboxylation of pyruvic acid in basic aqueous solution (pH 8.8). Acetoin is the final product of the reaction. Breslow [66] later showed that the hydrogen in position 2 of the thiazole ring of the coenzyme is exchanged with deuterium when deuterium oxide (D2O) is added to the incubation system. Thus, carbon 2 of the thiamine appears to react in this chemical process. The hydrogen in position 2 is acidic and is thus readily ionized to an anion in basic media (see Fig. 4-7). On the basis of these findings, researchers have proposed the following sequence of reactions to explain the catalytic action of carboxylase. The carbon 2 of the thiazo-... 

Why are B vitamins necessary If we consider the action of B vitamins as coenzymes, the answer is clear. There is no electrophilic functional group in the 20 amino acids of proteins. Coenzymes are therefore required for enzymes to carry out electrophilic and radical catalysis. Thiamin diphosphate is by itself a... 

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 thiazolium ylide is the intermediate in the action of thiamine pyrophosphate as a coenzyme it is intellectually related to cyanide ion, and just like cyanide it is able to catalyze the benzoin condensation. However, later there were assertions in the literature that the... 

In view of the fact that lipoic acid is a cyclic disulfide, it is tempting to speculate on its function as a 2-carbon carrier in pyruvate oxidation through a thioester in a fashion similar to the CoA fimction in this reaction. Similarly the disulfide nature of this factor invites speculation on its possible role as an oxidation-reduction coenzyme by going through a sulfhydryl form. It is a curious fact that three of the factors involved in pyruvate oxidation, thiamine pyrophosphate, CoA, and POF, all contain sulfur. In the first two of these factors, the sulfur appears to play an important role in their mechanism of action. By analogy one would suppose that the disulfide grouping of POF will be prominent in its mechanism of action. 

Recently Reed and DeBusk have isolated from natural materials a bound form of lipoic acid which seems to be somewhat closer to the coenzyme form. Analysis of this compound as well as its preparation from synthetic materials indicates that it is the amide of thiamine and lipoic acid (lipothiamide). This important discovery may eventually cast some light upon the mechanism of action of lipoic acid. It is significant that lipothiamide can restore pyruvate oxidation to deficient cells, and the authors conclude that lipothiamide may be part of the coenzyme for the oxidative decarboxylation of a-keto acids. 

Following absorption, thiamin is transported to the liver where it is phosphorylated under the action of ATP to form the coenzyme thiamin diphosphate (formerly called thiamin pyrophosphate or cocarboxylase), (see Fig. T-9) although this phosphorylation occurs rapidly in the liver, it is noteworthy that all nucleated cells appear to be capable of bringing about this conversion. 

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