title: Replacement of lysine 234 affects transition state stabilization in the active site of beta-lactamase TEM1.
abstract: Lysine 234 is a residue highly conserved in all beta-lactamases, except in the carbenicillin-hydrolyzing enzymes, in which it is replaced by an arginine. Informational suppression has been used to create amino acid substitutions at this position in the broad spectrum Escherichia coli beta-lactamase TEM-1, in order to elucidate the role of this residue which lies on the wall at the closed end of the active site cavity. The mutants K234R and K234T were constructed and their kinetic constants measured. Replacement of lysine 234 by arginine yields an enzyme with similar activity toward cephalosporins and most penicillins, except toward the carboxypenicillins for which the presence of the guanidine group enhances the transition state binding. The removal of the basic group in the mutant K234T yields a protein variant which retains a low activity toward penicillins, but losts drastically its ability to hydrolyze cephalosporins. Moreover, these two mutations largely decreased the affinity of the enzyme for penicillins (10-fold for K234R and 50-fold for K234T). This can be correlated with the disruption of the predicted electrostatic binding between the C3 carboxylic group of penicillins and the amine function of the lysine. Therefore, lysine 234 in the E. coli beta-lactamase TEM-1 is involved both in the initial recognition of the substrate and in transition state stabilization.
authors.affiliation: Institut National des Sciences Appliqueés, Centre National de la Recherche Scientifique URA 544, Toulouse, France.
authors.forename: F
authors.initials: F
authors.lastname: Lenfant
authors.forename: R
authors.initials: R
authors.lastname: Labia
authors.forename: J M
authors.initials: JM
authors.lastname: Masson
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D019818:Clavulanic Acid; D002969:Clavulanic Acids / Q000378:metabolism; D004269:DNA, Bacterial; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D002969:Clavulanic Acids; D004269:DNA, Bacterial; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; D019818:Clavulanic Acid; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D008239:Lysine