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

Abstract

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.

Glutamate 636 of the Escherichia coli Pyruvate Dehydrogenase-E1 Participates in Active Center Communication and Behaves as an Engineered Acetolactate Synthase with Unusual Stereoselectivity

“…Although AHAS and YPDC form the (S)-acetolactate configuration, PDHc-E1 forms the (R) configuration. The acetoin formed by PDHc-E1 also had the opposite configuration to that produced by the E477Q YPDC variant (26). As depicted in Fig.…”

“…A similar communication path was also suggested for E. coli PDHc-E1; however, the glutamate 636 is not located directly on this path (22) (16). For comparison, the D28A variant of YPDC produced (S)-acetolactate with a k cat ϭ 0.052 s Ϫ1 (26). According to the CD experiments, the acetolactate produced in the PDHc reaction is optically active, as was the case with the D28A variant of YPDC (26) and AHAS (28).…”

“…Also, as with YPDC, it appears that it is the negative charge on these residues which helps to avoid the side reaction by electrostatic repulsion vis à vis the second molecule of pyruvate, because with both YPDC and PDHc-E1, conversion of the aspartate (YPDC) or glutamate (PDHc-E1) to the amidated form or to alanine led to the same outcome. It is interesting to speculate why on benzoylformate decarboxylase, an enzyme analogous to YPDC, where the methyl group of pyruvate is replaced by a phenyl ring, the residue Ser 26 occupies the position corresponding to Asp 28 of YPDC (30,31). Perhaps, although the YPDC and PDHc-E1 require the negative charge at Asp 28 and Glu 636 , respectively, to repel the second pyruvate, on BFD the likelihood of this side reaction is diminished because of the much larger size of the substrate benzoylformate compared with pyruvate.…”

Saturation mutagenesis of putative catalytic residues of benzoylformate decarboxylase provides a challenge to the accepted mechanism

“…In the unliganded enzyme His-70 interacts with Glu-28 and, through a water molecule, with Ser-26 and the C2 position of ThDP (3,22). When (R)-mandelate binds, the water molecule is displaced with little other change in the position of the active site residues (4).…”

Characterization of Phenylpyruvate Decarboxylase, Involved in Auxin Production of Azospirillum brasilense

“…Glu468 is a conserved catalytic residue in pyruvate decarboxylase and IPDC Ec , which has been proposed to be involved in both preand postdecarboxylation steps. More specifically a role in protonation of the enamine-carbanion intermediate has been proposed (20,26,32). Enzymes with amino acid substitutions in this position showed an impaired release of the aldehyde product.…”