title: Probing the molecular basis of substrate specificity, stereospecificity, and catalysis in the class II pyruvate aldolase, BphI.
abstract: 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 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.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Ontario, Canada.
authors.forename: Perrin
authors.initials: P
authors.lastname: Baker
authors.forename: Jason
authors.initials: J
authors.lastname: Carere
authors.forename: Stephen Y K
authors.initials: SY
authors.lastname: Seah
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D007651:Keto Acids / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics; D019289:Pyruvic Acid / Q000737:chemistry*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D007651:Keto Acids; D019289:Pyruvic Acid; D007652:Oxo-Acid-Lyases