Catalytic acid-base groups in yeast pyruvate decarboxylase. 2. Insights into the specific roles of D28 and E477 from the rates and stereospecificity of formation of carboligase side products

Yeast pyruvate decarboxylase (YPDC), in addition to forming its metabolic p roduct acetaldehyde, can also carry out carboligase reactions in which the central enamine intermediate reacts with acetaldehyde or pyruvate (instead of the usual proton electrophile), resulting in the formation of acetoin an d acetolactate, respectively (typically, 1% of the total reaction). Due to the common mechanism shared by the acetaldehyde-forming and carboligase rea ctions through decarboxylation, a detailed analysis of the rates and stereo chemistry of the carboligase products formed by the E4774, D28A, and D28N a ctive center YPDC variants was undertaken. While substitution at either pos ition led to an approximately 2-3 orders of magnitude lower catalytic effic iency in acetaldehyde formation, the rate of acetoin formation by the E477Q and D28N variants was higher than that by wild-type enzyme. Comparison of the steady-state data for acetaldehyde and acetoin formation revealed that the rate-limiting step for acetaldehyde formation by the D28A, H114F, H115F , and E4774 variants is a step post-decarboxylation. In contrast to the wil d-type YPDC and the E4774 variant, the D28A and D28N variants could synthes ize acetolactate as a major product. The lower overall rate of side-product formation by the D28A variant than wild-type enzyme attests to participati on of D28 in steps leading up to and including decarboxylation. The results also provide insight into the state of ionization of the side chains exami ned. (R)-Acetoin is produced by the variants with greater enantiomeric exce ss than by wild-type YPDC. (S)-Acetolactate is the predominant enantiomer p roduced by the D28 substituted variants, the same configuration as produced by the related plant acetolactate synthase.