title: The mutation Lys234His yields a class A beta-lactamase with a novel pH-dependence.
abstract: The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.
authors.affiliation: University of Sussex, School of Biological Sciences, Falmer, Brighton, U.K.
authors.forename: J
authors.initials: J
authors.lastname: Brannigan
authors.forename: A
authors.initials: A
authors.lastname: Matagne
authors.forename: F
authors.initials: F
authors.lastname: Jacob
authors.forename: C
authors.initials: C
authors.lastname: Damblon
authors.forename: B
authors.initials: B
authors.lastname: Joris
authors.forename: D
authors.initials: D
authors.lastname: Klein
authors.forename: B G
authors.initials: BG
authors.lastname: Spratt
authors.forename: J M
authors.initials: JM
authors.lastname: Frère
mesh_terms: D000667:Ampicillin / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004795:Enzyme Stability; D015870:Gene Expression; D006639:Histidine; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine; D009154:Mutation; D010400:Penicillin G / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D006639:Histidine; D000667:Ampicillin; D001618:beta-Lactamases; D008239:Lysine; D010400:Penicillin G