title: The role of the non-conserved residue at position 104 of class A beta-lactamases in susceptibility to mechanism-based inhibitors.
abstract: The role of the non-conserved amino acid residue at position 104 of the class A beta-lactamases, which comprises a highly conserved sequence of amino acids at the active sites of these enzymes, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace Asp104 with the corresponding Staphylococcus aureus PC1 residue Ala104. Kinetic data obtained with the purified Asp104Ala B. cereus 569/H beta-lactamase I was compared to that obtained from the wild-type B. cereus and S. aureus enzymes. Replacement of amino acid residue 104 had little effect on the Michaelis parameters for the hydrolysis of both S- and A-type penicillins. Relative to wild-type enzyme, the Asp104Ala beta-lactamase I had 2-fold higher Km values for benzylpenicillin and methicillin, but negligible difference in Km for ampicillin and oxacillin. However, kcat values were also slightly increased resulting in little change in catalytic efficiency, kcat/Km. In contrast, the Asp104Ala beta-lactamase I became more like the S. aureus enzyme in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amido-penicillanic acid sulfone with respect to both response to the inhibitors and subsequent enzymatic properties. Based on the known three-dimensional structures of the Bacillus licheniformis 749/C, Escherichia coli TEM and S. aureus PC1 beta-lactamases, a model for the role of the non-conserved residue at position 104 in the process of inactivation by mechanism-based inhibitors is proposed.
authors: Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry, University of Waterloo, Waterloo, ON N2L 3G1, Canada.
authors: F
authors: F
authors: Guo
authors: J
authors: J
authors: Huynh
authors: G I
authors: GI
authors: Dmitrienko
authors: T
authors: T
authors: Viswanatha
authors: A J
authors: AJ
authors: Clarke
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001409:Bacillus cereus / Q000201:enzymology; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D013211:Staphylococcus aureus / Q000201:enzymology; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000096:biosynthesis / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D001224:Aspartic Acid; D001618:beta-Lactamases; D000409:Alanine