Probing the Molecular Basis of Substrate Specificity, Stereospecificity, and Catalysis in the Class II Pyruvate Aldolase, Bphl

BphI, a pyruvate-specific class H aldolase found in the polychlorinated biphenyls (PCBs) degradation pathway, catalyzes the reversible C C bond cleavage of (4S)-hydroxy-2-oxoacids to form pyruvate and an aldehyde.. Mutations were. introduced into bphI to probe the contribution of active site residues to substrate recognition and catalysis. In contrast to the wild-type enzyme that has similar specificities for acetaldehyde and propionaldehyde, the. L87A variant exhibited a 40-fold preference for propionaldehyde over acetaldehyde.,The, specificity Constant of the L89A variant in the aldol addition reaction using pentaldehyde is, increased similar to 50-fold; making it more catalytically efficient for pentaldehyde utilization compared to the wild-type utilization of the natural substrate, acetaldehyde. Replacement of Tyr-290 with phenylalanine or serine resulted in a loss of stereochemical control as the variants were able to utilize substrates with both R and S:configurations at C4 with similar kinetic parameters. Aldol cleavage and pyruvate alpha-proton exchange activity were undetectable in the R16A variant, supporting the role of Arg-16 in stabilizing a pyruvate enolate intermediate. The pH :dependence of the enzyme is consistent with a single deprotonation by a catalytic base with pK(a) values of approximately 7. In H20A, and H20S variants, pH profiles show the dependence of enzyme activity on hydroxide concentration. On the basis of these results, a catalytic mechanism is proposed.