Site-directed mutagenesis on TEM-1 beta-lactamase: role of Glu166 in catalysis and substrate binding.
Class A beta-lactamases are the major cause of bacterial resistance to beta-lactam antibiotics. In these active-site serine hydrolases, glutamic acid 166 has been hypothesized to act as a general acid-base catalyst. Replacing this residue by tyrosine in TEM-1 beta-lactamase yields an enzyme the activity of which is substantially lowered and strongly dependent on pH, thus confirming the alleged role of Glu166 in catalysis. This substitution also resulted in a spectacular change in substrate profile, the mutant enzyme being more active on cephalosporins than on penicillins. In fact, the E166Y enzyme behaves much like a class C enzyme, with high affinity and low hydrolytic activity towards second and third generation cephalosporins. Glu166 therefore seems to play a major part in defining the substrate profile of class A beta-lactamases.
Delaire
M
M
INSA, Laboratoire de Génie Biochimique, UA 544 du CNRS, Toulouse, France.
Lenfant
F
F
Labia
R
R
Masson
J M
JM
D001665:Binding Sites / Q000235:genetics; D002511:Cephalosporins / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D016153:Genes, Suppressor; D016297:Mutagenesis, Site-Directed; D010403:Penicillin Resistance / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000235:genetics*
D002511:Cephalosporins; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1