Probing the molecular basis of substrate specificity, stereospecificity, and catalysis in the class II pyruvate aldolase, BphI.

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