title: Role of asparagine 152 in catalysis of beta-lactam hydrolysis by Escherichia coli AmpC beta-lactamase studied by site-directed mutagenesis.
abstract: The role of asparagine 152 in the catalytic mechanism of Escherichia coli AmpC beta-lactamase has been investigated by site-directed mutagenesis. The residue has been replaced by aspartic acid, glutamic acid, histidine, and leucine. All the substitutions had similar effects on the activity toward substrates and inhibitors. The rate of substrate hydrolysis decreased by factors of 500-5000. The rates of both acylation (2-50-fold decrease) and deacylation (50-500-fold decrease) were affected, indicating a role for Asn152 in both processes. The wild-type AmpC beta-lactamase appears to exist as an equilibrium mixture of two forms, identified by their different kinetic properties. The Asn152 mutations affected the activity of the slow-reacting form much more than that of the fast-reacting form, but they did not appear to affect the interconversion of these two kinetic forms. Comparison of these observations with results obtained with mutation of the equivalent residues in other classes of penicillin-sensitive enzyme indicates that there are quite profound differences between the catalytic mechanisms of these enzymes despite a high degree of conservation of amino acids in the active center, and of the overall three-dimensional structure.
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
authors.forename: A
authors.initials: A
authors.lastname: Dubus
authors.forename: S
authors.initials: S
authors.lastname: Normark
authors.forename: M
authors.initials: M
authors.lastname: Kania
authors.forename: M G
authors.initials: MG
authors.lastname: Page
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism; D001216:Asparagine / Q000737:chemistry*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism; D047090:beta-Lactams
chemical_list: D000900:Anti-Bacterial Agents; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; D001216:Asparagine; D001618:beta-Lactamases