PYRUVATE DECARBOXYLASE - A MOLECULAR MODELING STUDY OF PYRUVATE DECARBOXYLATION AND ACYLOIN FORMATION

Using crystal structure data for the pyruvate decarboxylase from Sacch aromyces uvarum (which is nearly identical with the enzyme from Saccha romyces cerevisiae), molecular modeling studies have been carried out to investigate the mode of action of the enzyme. Each step of the deca rboxylation mechanism can be explained by assuming that the 4'-amino g roup of thiamin diphosphate (TDP) acts as a general acid and, in its d eprotonated form, as a general base. The carboxyl group of Glu 477 pla ys a key role in both pyruvate decarboxylation and acyloin formation. In the first case it interacts with the carboxylate group of pyruvate to stabilize the incipient dianion formed by attack of the thiazolium carbanion on pyruvate. In the second case, it interacts with the devel oping alkoxide anion arising from attack on acetaldehyde or benzaldehy de by the carbanion-enamine intermediate. These studies have permitted the assignment of configuration to all of the key intermediates in th e catalytic process. Thus the carbanion-enamine intermediate 5 is foun d to have the E-configuration. The S-configuration is imposed on the 2 -(2-hydroxypropionyl)ethyl)thiamin diphosphate intermediate 4 by the c hiral conformation induced in the achiral cofactor through its interac tions with the protein. The R-configuration is assigned to the 2-(1-hy droxyethyl)thiamin diphosphate intermediate 6 arising through protonat ion of the carbanion-enamine intermediate 5. The tight stereochemical control observed in acyloin formation from aromatic aldehydes and pyru vate is explained, as is the relaxed stereocontrol in acyloin formatio n from acetaldehyde and pyruvate.