CLINICAL INHIBITOR-RESISTANT MUTANTS OF THE BETA-LACTAMASE TEM-1 AT AMINO-ACID POSITION-69 - KINETIC-ANALYSIS AND MOLECULAR MODELING

The kinetic parameters of three IRT (Inhibitor-Resistant-TEM-derived-) beta-lactamases (IRT-5, IRT-6 and IRT-I69) were determined for substr ates and the beta-lactamase inhibitors: clavulanic acid, sulbactam and tazobactam. and compared with those of TEM-1 beta-lactamase. The cata lytic behaviour of the beta-lactamases towards substrates and inhibito rs was correlated with the properties of the amino acid at position AB L69. The three IRT beta-lactamases contain at that position a residue Ile, Leu and Val, amino acids whose side-chain are branched. Molecular modelling shows that the methyl groups of Ile-69 (C gamma 2) and Val- 69 (C gamma 1) produced steric constraints with the side chain of Asn- 170 as well as the main chain nitrogen of Ser-70. a residue contributi ng to the oxyanion hole. We suggest that hydrophobicity could be the m ain factor responsible for the kinetic properties of Met69Leu (IRT-5), as no steric effects could be detected by molecular modelling. Hydrop hobicity and steric constraints are combined in Met69Ile and Met69Val, IRT-I69 and IRT-6, respectively. (C) 1998 Elsevier Science B.V.