bi101947g_si_001.pdf (1.58 MB)
Probing the Molecular Basis of Substrate Specificity, Stereospecificity, and Catalysis in the Class II Pyruvate Aldolase, BphI
journal contributionposted on 2011-05-03, 00:00 authored by Perrin Baker, Jason Carere, Stephen Y. K. Seah
BphI, a pyruvate-specific class II 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 ∼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 α-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 pKa 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.
pH dependencesubstrate recognitionH 20S variantsAldol cleavagedegradation pathwayform pyruvatepyruvate enolatehydroxide concentrationS configurationspH profiles showL 89A variantL 87A variantMolecular BasisC 4propionaldehydesite residuesClass II Pyruvate AldolaseH 20Aaldol addition reactionenzyme activityR 16A variantpentaldehyde utilizationpKa valuesacetaldehydeSubstrate Specificitypolychlorinated biphenylsstereochemical control