SITE-DIRECTED MUTAGENESIS OF GLUTAMATE-166 IN 2 BETA-LACTAMASES - KINETIC AND MOLECULAR MODELING STUDIES

The catalytic pathway of class A beta-lactamases involves an acyl-enzy me intermediate where the substrate is ester-linked to the Ser-70 resi due, 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 replacem ent 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 a cyl-enzymes with beta-lactam antibiotics, Although acylation of the mo dified proteins by benzylpenicillin remained relatively fast, it was s ignificantly impaired when compared to that observed with the wild-typ e enzyme. Moreover, the E166N substitution resulted in a spectacular m odification of the substrate profile much larger than that described f or 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 o f Glu-166 and of the ''catalytic'' mater molecule in both the acylatio n and the deacylation processes.