Article (Scientific journals)
Site-directed mutagenesis of glutamate 166 in two beta-lactamases. Kinetic and molecular modeling studies.
Guillaume, Gilliane; Vanhove, M; Lamotte-Brasseur, Josette et al.
1997In Journal of Biological Chemistry, 272 (9), p. 5438-44
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Keywords :
Acylation; Cefoxitin/metabolism; Cefuroxime/metabolism; Cephaloridine/metabolism; Enzyme Stability; Glutamic Acid/genetics; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Penicillin G/pharmacology; Streptomyces; beta-Lactamases/genetics
Abstract :
[en] 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Guillaume, Gilliane
Vanhove, M
Lamotte-Brasseur, Josette 
Ledent, P
Jamin, M
Joris, Bernard ;  Université de Liège - ULiège > Département des sciences de la vie > Physiologie et génétique bactériennes
Frère, Jean-Marie ;  Université de Liège - ULiège > Centre d'ingénierie des protéines
Language :
English
Title :
Site-directed mutagenesis of glutamate 166 in two beta-lactamases. Kinetic and molecular modeling studies.
Publication date :
1997
Journal title :
Journal of Biological Chemistry
ISSN :
0021-9258
eISSN :
1083-351X
Publisher :
American Society for Biochemistry and Molecular Biology, Baltimore, United States - Maryland
Volume :
272
Issue :
9
Pages :
5438-44
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 22 May 2012

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