Thiamin Pyrophosphate-Dependent Reactions

The decarboxylation of pymvic acid to yield carbon dioxide and a two-carbon fragment is catalyzed by a class of enzymes that require thiamin pyrophosphate (TPP) and a divalent metal ion. We will restrict our attention to the simplest member of the class, pyruvate decarboxylase 132, 133), which catalyzes reaction 18). 

This is the most studied TPP-dependent enzyme, and it is likely that mechanisms of other thiamin pyrophosphate-dependent decarboxylations are similar. Editor s note Other aspects of thiamin pyrophosphate chemistry are presented in Chapter 7 by Kluger.) 

An understanding of mechanisms of thiamin pyrophosphate-dependent processes must begin with the classic work of Breslow 105, 134), who showed that the hydrogen at C-2 of thiamin pyrophosphate can be removed by bases and that the resulting anion is highly reactive. The at this site is 18 135). In an enzyme active site, this p a value may be considerably lower, as the value decreases with decreasing medium polarity. Reaction of the anion with a variety of carbonyl compounds (e.g., acetaldehyde, pyruvate) gives rise to characterizable adducts 132). 

Following decarboxylation, the two-carbon-TPP adduct is protonated. This compound has also been synthesized and studied (132) interestingly, it does not release acetaldehyde in nonenzymic reactions, whereas it obviously does in the enzymic reaction (132). Conformational control on the enzyme may be responsible. Consistent with this, glyoxylic acid is decarboxylated by pyruvate decarboxylase, but the product, hydroxymethyl-TPP, does not release formaldehyde from the enzyme (132). 

As in the case of pyridoxal phosphate, the key to reaction in this case is the use of a heterocyclic compound as an electron sink in the decarboxylation step. Conformational control of the TPP-pyruvate adduct may also be important. The enzyme active site is probably nonpolar, and this provides a significant catalytic factor (112). 

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This thiamin pyrophosphate-dependent enzyme [EC 4.1.2.9] catalyzes the reaction of D-xylulose 5-phosphate with orthophosphate to produce acetyl phosphate, d-glyceraldehyde 3-phosphate, and water. 

This thiamin pyrophosphate-dependent enzyme [EC 2.2.1.1], also known as glycolaldehyde transferase, catalyzes the reversible reaction of sedoheptulose 7-phos-phate with D-glyceraldehyde 3-phosphate to produce D-ribose 5-phosphate and o-xylulose 5-phosphate. The enzyme exhibits a wide specificity for both reactants. It also can catalyze the reaction of hydroxypyruvate with R—CHO to produce carbon dioxide and R—CH(OH)—C(=0)—CH2OH. Transketolase isolated from Alkaligenes faecalis shows high activity with D-erythrose as the acceptor substrate. 

NADP+ differs from NAD+ only by phosphorylation of the C-2 OH group on the adenosyl moiety. The redox potentials differ only by about 5 mV. Why do you suppose it is necessary for the cell to employ two such similar redox cofactors Thiamine-pyrophosphate-dependent enzymes catalyze the reactions shown below. Write a chemical mechanism that shows the catalytic role of the coenzyme, (a) O O... 

The quinone ring is derived from isochorismic acid, formed by isomerization of chorismic acid, an intermediate in the shikirnic acid pathway for synthesis of the aromatic amino acids. The first intermediate unique to menaquinone formation is o-succinyl benzoate, which is formed by a thiamin pyrophosphate-dependent condensation between 2-oxoglutarate and chorismic acid. The reaction catalyzed by o-succinylbenzoate synthetase is a complex one, involving initially the formation of the succinic semialdehyde-thiamin diphosphate complex by decarboxylation of 2-oxoglutarate, then addition of the succinyl moiety to isochorismate, followed by removal of the pyruvoyl side chain and the hydroxyl group of isochorismate. 

These two 5-C sugars, R-5-P and Xu-5-P, are now interconverted to a 7-C sugar, Sedoheptulose-7-P, and a 3-C sugar, Glyceraldehyde-3-P. This reaction is catalyzed by Transketolase, a Thiamine pyrophosphate dependent enz)rme which catalyzes the transfer of C2 units. In the first part of this reaction the TPP carbanion makes a nucleophilic attack on the carbonyl group of xylulose. In the resulting intermediate the C2-C3 bond is destabilized and cleavage takes place to yield the enzyme bound 2-(l,2-dihydroxyethyl)-TPP resonance stabilized carbanion ... 

Demir AS, Ayhan P, Sopaci SB. Thiamine pyrophosphate dependent enzyme catalyzed reactions Stereoselective C—C bond formations in water. CLEAN-Soil, Air, Water 2007 35 (5) 406 12. 

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... 

Acyloins (a-hydroxy ketones) are formed enzymatically by a mechanism similar to the classical benzoin condensation. The enzymes that can catalyze reactions of this type arc thiamine dependent. In this sense, the cofactor thiamine pyrophosphate may be regarded as a natural- equivalent of the cyanide catalyst needed for the umpolung step in benzoin condensations. Thus, a suitable carbonyl compound (a -synthon) reacts with thiamine pyrophosphate to form an enzyme-substrate complex that subsequently cleaves to the corresponding a-carbanion (d1-synthon). The latter adds to a carbonyl group resulting in an a-hydroxy ketone after elimination of thiamine pyrophosphate. Stereoselectivity of the addition step (i.e., addition to the Stand Re-face of the carbonyl group, respectively) is achieved by adjustment of a preferred active center conformation. A detailed discussion of the mechanisms involved in thiamine-dependent enzymes, as well as a comparison of the structural similarities, is found in references 1 -4. 

Intermediates of this type have the necessary chemical reactivity for cleaving the bonds indicated in figure 10.1b and c. The decarboxylated product of the pyruvate adduct shown in equation (2) is resonance-stabilized by the thiazolium ring (fig. 10.2a). This intermediate may be protonated to a-hydroxyethyl thiamine pyrophosphate (fig. I0.2d) alternatively, it may react with other electrophiles, such as the carbonyl groups of acetaldehyde or pyruvate, to form the species in figure 10.2b and c or it may be oxidized to acetyl-thiamine pyrophosphate (fig. 10.2e). The fate of the intermediate depends on the reaction specificity of the enzyme with which the coenzyme is associated. 

The a-keto acid decarboxylases such as pyruvate (E.C. 4.1.1.1) and benzoyl formate (E.C. 4.1.1.7) decarboxylases are a thiamine pyrophosphate (TPP)-dependent group of enzymes, which in addition to nonoxidatively decarboxylating their substrates, catalyze a carboligation reaction forming a C-C bond leading to the formation of a-hydroxy ketones.269-270 The hydroxy ketone (R)-phenylacetylcarbinol (55), a precursor to L-ephedrine (56), has been synthesized with pyruvate decarboxylase (Scheme 19.35). BASF scientists have made mutations in the pyruvate decarboxylase from Zymomonas mobilis to make the enzyme more resistant than the wild-type enzyme to inactivation by acetaldehyde for the preparation of chiral phenylacetylcarbinols.271... 

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. 

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 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. 

The decarboxylation of pyruvic acid is an example of a more general type of biochemical reaction the decarboxylation of a-keto acids. The reaction is complex and occurs in several consecutive steps. The intermediates have been identified, but little is known of the enzymes involved. The reaction starts with the complexion of pyruvic acid with one molecule of enzyme-bound thiamine pyrophosphate. This is followed by decarboxylation of pyruvic acid and the formation of an intermediate, 2-acetylthiamine pyrophosphate, in which the aldehyde carbon of the acetyl is bound to the carbon 2 of the thiozole ring of the thiamine pyrophosphate. In the second step, the aldehyde is oxidized, the disulfide bond of enzyme-bound lipoic acid is reduced, and the free enzyme-bound thiamine pyrophosphate is restored. The tWrd step of the reaction involves the transacylation from reduced lipoic acid to CoA. Finally, lipoic acid is reoxidized by the catalytic activity of an NAD-dependent flavoprotein, lipoic dehydrogenase (see Fig. 1-14). 

Sodium l,l-dimethoxyethyl(methyl)phosphinate 2 was found to be the most effective herbicidal compound among plant PDHc El inhibitors by Baillie et al. s work. 2 was presumably hydrolyzed to sodium salt of acetyl(methyl)phosphinic acid 1-2 in vivo to exhibit herbicidal activity (Scheme 4.10). 1-2 displayed higher enzyme inhibition and herbicidal activity than 1-1. It has been found that 1-1 was a competitive inhibitor of PDHc, but 1-2 caused time-dependent inhibition. Baillie et al. gave a possible explanation for this result, the initial binding of inhibitors to the pyruvate site and subsequent reaction with thiamine pyrophosphate were rapid and reversible for both 1-1 and 1-2. In the case of 1-2, an enzyme-inhibitor complex was first formed and then underwent a time-dependent, essentially irreversible transformation to produce a more tightly bound form. In other words, 1-2 could act as a slow, tight binding inhibitor [1]. 

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