Thiamine-pyrophosphate

Thiamine pyrophosphate (TPP) is the active form of thiamine in the cells of all organisms and, as such, is important as a coenzyme in the decarboxylation of a-keto acids, as well as in the biosynthesis of certain acyloins. In view of the recent findings of Reed and De Busk (see section on pyruvate oxidation factor) TPP may form a complex with a-lipoic acid, which may be the coenzyme for the oxidative decarboxylation of a-keto acids. 

Although thiamine was isolated in pure form in 1926, the structure and 

Although Barron had shown as early as 1932 that gonococci could carry out an oxidative breakdown of pyruvate to acetate and CO2, it was the work of Peters and his group on thiamine-deficient rats and pigeons which indicated that thiamine was necessary for the oxidative breakdown of pyruvate which occurred in animal tissues. Shortly after it was shown by many investigators that pyruvate breakdown in animal tissues, as well as in many bacteria, can occur only in the presence of suitable hydrogen acceptors to yield acetate and CO2 and that free acetaldehyde was not an intermediate. 

Lipmann s discovery in 1939 that acetyl phosphate results from the oxidative decarboxylation of pyruvate foretold the fundamental role of TPP in initiating reactions leading to the production of an activated 2-car-bon fragment. 

TPP is inactivated by treatment with 1 N hydrochloric acid at 100° for 15 min. owing to the hydrolysis of the pyrophosphate linkage and yields orthophosphate and thiamine monophosphate. Treatment of TPP with alkali splits off pyrophosphate and gives free thiamine. TPP, like thiamine, 

The role of thiamine pyrophosphate appears to be the formation of compounds possessing structures analogous to those of the beta-keto 

As shown in Fig. 7, the first step involved in the decarboxylation of an alpha-keto acid by thiamine pyrophosphate is the addition of the carbonyl by the carbanion nucleophile, followed by decarboxylation of the acid. As reviewed by Jencks (1975), investigations of the role of thiamine pyrophosphate analogs in the pyruvate dehydrogenase-catalyzed decarboxylation of pyruvate have provided evidence for rate enhancement by a desolvation effect. This effect comes about because of the removal of both the carboxylate group of the substrate and the cationic nitrogen of the coenzyme from an aqueous environment to the hydrophobic active site of the enzyme. The bulky and apparently chemically nonfunctional pyrophosphate and pyrimidine moieties of the 

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Thiamin pyrophosphate- natures acyl anion equivalent for trans ketolization reactions... 

In this thiamine pyrophosphate-mediated process, ben2aldehyde (29), added to fermenting yeast, reacts with acetaldehyde (qv) (30), generated from glucose by the biocatalyst, to yield (R)-l-phen5l-l-hydroxy-2-propanone (31). The en2ymatically induced chiral center of (31) helps in the asymmetric reductive (chemical) condensation with methylamine to yield (lR,23)-ephedrine [299-42-3] (32). Substituted ben2aldehyde derivatives react in the same manner (80). 

The pathways for thiamine biosynthesis have been elucidated only partiy. Thiamine pyrophosphate is made universally from the precursors 4-amino-5-hydroxymethyl-2-methylpytimidinepyrophosphate [841-01-0] (47) and 4-methyl-5-(2-hydroxyethyl)thiazolephosphate [3269-79-2] (48), but there appear to be different pathways ia the eadier steps. In bacteria, the early steps of the pyrimidine biosynthesis are same as those of purine nucleotide biosynthesis, 5-Aminoimidazole ribotide [41535-66-4] (AIR) (49) appears to be the sole and last common iatermediate ultimately the elements are suppHed by glycine, formate, and ribose. AIR is rearranged in a complex manner to the pyrimidine by an as-yet undetermined mechanism. In yeasts, the pathway to the pyrimidine is less well understood and maybe different (74—83) (Fig. 9). 

The TK-catalyzed reaction requires the presence of thiamine pyrophosphate and Mg " as cofactors. Although the substrate specificity of the enzyme has not been thoroughly investigated, it has been shown that the enzyme accepts a wide variety of 2-hydroxyaldehydes including D-glyceraldehyde 3-phosphate [591-57-1], D-glyceraldehyde [453-17-8], D-ribose 5-phosphate /47(9(9-2%/7, D-erythrose 4-phosphate and D-erythrose [583-50-6] (139,149—151). 

As shown in Eigure 18.17, thiamine is composed of a substituted thiazole ring joined to a substituted pyrimidine by a methylene bridge. It is the precursor of thiamine pyrophosphate (TPP), a coenzyme involved in reactions of carbo-... 

Water-Soluble Thiamine (vitamin Bj) Thiamine pyrophosphate... 

FIGURE 18.17 Thiamine pyrophosphate (TPP), the active form of vitamin is formed by the action of TPP-synthetase. 

FIGURE 18.18 Thiamine pyrophosphate participates in (a) the decarboxylation of n-keto acids and (b) the formation and cleavage of n-hydroxyketones. 

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... 

This resonance-stabilized intermediate can be protonated to give hydroxyethyl-TPP. This well-characterized intermediate was once thought to be so unstable that it could not be synthesized or isolated. However, its synthesis and isolation are actually routine. (In fact, a substantial amount of the thiamine pyrophosphate in living things exists as the hydroxyethyl form.)... 

Based on the action of thiamine pyrophosphate in catalysis of the pyruvate dehydrogenase reaction, suggest a suitable chemical mechanism for the pyruvate decarboxylase reaction in yeast ... 

Stepl of Figure 29.11 Addition of Thiamin Diphosphate The conversion of pyruvate to acetyl CoA begins by reaction of pyruvate with thiamin diphosphate, a derivative of vitamin B(. Formerly called thiamin pyrophosphate, thiamin diphosphate is usually abbreviated as TPP. The spelling thiamine is also correct and frequently used. 

A number of lyases are known which, unlike the aldolases, require thiamine pyrophosphate as a cofactor in the transfer of acyl anion equivalents, but mechanistically act via enolate-type additions. The commercially available transketolase (EC 2.2.1.1) stems from the pentose phosphate pathway where it catalyzes the transfer of a hydroxyacetyl fragment from a ketose phosphate to an aldehyde phosphate. For synthetic purposes, the donor component can be replaced by hydroxypyruvate, which forms the reactive intermediate by an irreversible, spontaneous decarboxylation. 

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. 

Fermenting baker s yeast also catalyzes the 1,4-addition of a formal trifluoroethanol-d1-synthon to a,/i-unsaturated aldehydes, to give optically active l,l,l-trifluoro-2-hydroxy-5-alka-nones52. Presumably, the mechanism involves oxidation of the alcohol to the corresponding aldehyde followed by an umpolung step with thiamine pyrophosphate and Michael addition to the a,/i-unsaturated aldehyde. For example, l,l,l-trifluoro-2-hydroxy-5-hexanone (yield 26%, ee 93%) is thus obtained from trifluoroethanol and l-bnten-3-one. 

Scheme 2.—Phosphorylations and condensation in the biosynthesis of thiamine pyrophosphate.

GA-3-P = glyceraldehyde 3-phosphate TPP = thiamine pyrophosphate DOXP = 1-deoxy-D-syiuiose CTP = oytosine triphosphate ATP = adenosine triphosphate ... 

These enzymes catalyse the non-hydrolytic cleavage of bonds in a substrate to remove specific functional groups. Examples include decarboxylases, which remove carboxylic acid groups as carbon dioxide, dehydrases, which remove water, and aldolases. The decarboxylation of pyruvic acid (10.60) to form acetaldehyde (10.61) takes place in the presence of pyruvic decarboxylase (Scheme 10.13), which requires the presence of thiamine pyrophosphate and magnesium ions for activity. 

A somewhat more trivial thing to remember about the HMP pathway is that this is one of the places you ve seen the vitamin thiamin pyrophosphate. This cofactor is necessary for the transketolase reaction that is in the middle of the HMP pathway. The transketolase reaction converts two C-5 sugars to a C-7 and a C-3. The other place you ve seen thiamin pyrophosphate as a cofactor is in the pyruvate dehydrogenase and a-ketoglutarate dehydrogenase reactions. 

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