Lactylthiamin

Kluger and Brandi (1986b) also studied the decarboxylation and base-catalysed elimination reactions of lactylthiamin, the adduct of pyruvate and thiamin (Scheme 2). These reactions are nonenzymic models for reactions of the intermediates formed during the reaction catalysed by the enzyme pyruvate decarboxylase. The secondary j3-deuterium KIE for the decarboxylation was found to be 1.09 at pH 3.8 in 0.5 mol dm-3 sodium acetate at 25°C. In the less polar medium, 38% ethanolic aqueous sodium acetate, chosen to mimic the nonpolar reactive site in the enzyme, the reaction is significantly faster but the KIE was, within experimental error, identical to the KIE found in water. This clearly demonstrates that the stabilization of the transition state by hyperconjugation is unaffected by the change in solvent. 

The adducts a-lactylthiamin and a-lactylthiamin diphosphate have both been synthesized.84 98 100 As long as a-lactylthiamin is kept as a dry solid or at low pH, it is stable. However, it decarboxylates readily in neutral solution (Eq. 14-21). Decarboxylation is much... 

The crystal structures of thiamin-dependent enzymes (see next section) as well as modeling102 103 suggest that lactylthiamin pyrophosphate has the conformation shown in Eq. 14-21. If so, it would be formed by the addition of the ylid to the carbonyl of pyruvate in accord with stereoelectronic principles, and the carboxylate group would also be in the correct orientation for elimination to form the enamine in Eq. 14-21, step b.82 83a A transient 380- to 440-nm absorption band arising during the action of pyruvate decarboxylase has been attributed to the enamine. 

There are significant differences in the reactivity of synthetic intermediate analogues for these reactions and the corresponding intermediates in the enzymic system. Lienhard and coworkers42,43 reported that the rate of decarboxylation of 2-(l-carboxy-l-hydroxyethyl)-3,4-dimethylthiazolium chloride is very fast relative to pyruvate (whose reaction is too slow to observe) but slower than the enzymic decarboxylation of pyruvate decarboxylase (PDC) by a factor of 105. Similar observations of a catalytic gap were seen for the rate of decarboxylation of lactylthiamin compared to PDC and the... 

The product, a-acetolactate, is formally derived from the acyl carbanion generated by the decarboxylation of pyruvate with a second molecule of pyruvate. The formation of lactylthiamin diphosphate on the enzyme generates initially the unprotonated adduct, which contains the enamine equivalent of a carbanion. This can add to the carbonyl group of a second molecule of pyruvate to form acetolactate (Scheme 35). 

ScHEME 35. Lactylthiamin diphosphate is converted to (hydroxyethyl)thiamin diphosphate. 

Indeed both -lactylthiamine pyrophosphate (XX) and a-hydroxyethyl-thiamine pyrophosphate (XXI) have been isolated and identified as products after incubation of pyruvate with a purified carboxylase preparation " . When [2- - C]pyruvate is used, the radioactivity is found in the thiazole part of the molecule after sulfite cleavage of XXL Acetaldehyde is formed from pyruvic acid by yeast carboxylase by enzymic cleavage of intermediate XXI, Uberating thiamine pyTophosphate . XXI has also been identified as intermediate in the formation of acetyl-coenzyme A from pyruvic acid by p3u uvic oxidase . The transketolase reaction has been shown to proceed via a gly-colaldehyde-enzyme intermediate here one may expect to find dihydroxy-ethylthiamine pyrophosphate as active glycol-aldehyde . Such experiments strongly support Breslow s concept of the reaction mechanism. 

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