Site-directed mutagenesis of glutamate 166 in two beta-lactamases - Kinetic and molecular modeling studies

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'' mater molecule in both the acylation and the deacylation processes.