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 product 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 and acetolactate, respectively (typically, 1% of the total reaction). Due to the common mechanism shared by the acetaldehyde-forming and carboligase reactions through decarboxylation, a detailed analysis of the rates and stereochemistry of the carboligase products formed by the E477Q, D28A, and D28N active center YPDC variants was undertaken. While substitution at either position led to an approximately 2-3 orders of magnitude lower catalytic efficiency 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 E477Q variants is a step post-decarboxylation. In contrast to the wild-type YPDC and the E477Q variant, the D28A and D28N variants could synthesize acetolactate as a major product. The lower overall rate of side-product formation by the D28A variant than wild-type enzyme attests to participation of D28 in steps leading up to and including decarboxylation. The results also provide insight into the state of ionization of the side chains examined. (R)-Acetoin is produced by the variants with greater enantiomeric excess than by wild-type YPDC. (S)-Acetolactate is the predominant enantiomer produced by the D28-substituted variants, the same configuration as produced by the related plant acetolactate synthase.
Sergienko
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Department of Chemistry and Program in Cellular and Molecular Biodynamics, Rutgers, The State University of New Jersey, Newark, New Jersey 07102, USA.
Jordan
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D000079:Acetaldehyde / Q000378:metabolism; D000093:Acetoin / Q000378:metabolism; D000095:Acetolactate Synthase / Q000378:metabolism*; D000409:Alanine / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D003653:Decarboxylation; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007773:Lactates / Q000378:metabolism; D011767:Pyruvate Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000235:genetics
D007773:Lactates; D011994:Recombinant Proteins; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; C006359:alpha-acetolactate; D019289:Pyruvic Acid; D000093:Acetoin; D000095:Acetolactate Synthase; D011767:Pyruvate Decarboxylase; D000079:Acetaldehyde; D000409:Alanine