title: Site-directed mutagenesis of glutamate 166 in two beta-lactamases. Kinetic and molecular modeling studies.
abstract: The catalytic pathway of class A beta-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been proposed as candidates for the role of general base in the activation of the serine OH group. The replacement of Glu-166 by an asparagine in the TEM-1 and by a histidine in the Streptomyces albus G beta-lactamases yielded enzymes forming stable acyl-enzymes with beta-lactam antibiotics. Although acylation of the modified proteins by benzylpenicillin remained relatively fast, it was significantly impaired when compared to that observed with the wild-type enzyme. Moreover, the E166N substitution resulted in a spectacular modification of the substrate profile much larger than that described for other mutations of Omega-loop residues. Molecular modeling studies indicate that the displacement of the catalytic water molecule can be related to this observation. These results confirm the crucial roles of Glu-166 and of the "catalytic" water molecule in both the acylation and the deacylation processes.
authors.affiliation: Centre d'Ingénierie des Protéines and Laboratoire d'Enzymologie, Université de Liège, Institut de Chimie B6, Sart-Tilman, B-4000 Liège, Belgium.
authors.forename: G
authors.initials: G
authors.lastname: Guillaume
authors.forename: M
authors.initials: M
authors.lastname: Vanhove
authors.forename: J
authors.initials: J
authors.lastname: Lamotte-Brasseur
authors.forename: P
authors.initials: P
authors.lastname: Ledent
authors.forename: M
authors.initials: M
authors.lastname: Jamin
authors.forename: B
authors.initials: B
authors.lastname: Joris
authors.forename: J M
authors.initials: JM
authors.lastname: Frère
mesh_terms: D000215:Acylation; D002440:Cefoxitin / Q000378:metabolism; D002444:Cefuroxime / Q000378:metabolism; D002509:Cephaloridine / Q000378:metabolism; D004795:Enzyme Stability; D018698:Glutamic Acid / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010400:Penicillin G / Q000494:pharmacology; D013302:Streptomyces; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D018698:Glutamic Acid; D002440:Cefoxitin; D001618:beta-Lactamases; D002509:Cephaloridine; D002444:Cefuroxime; D010400:Penicillin G