Clinical inhibitor-resistant mutants of the beta-lactamase TEM-1 at amino-acid position 69. Kinetic analysis and molecular modelling.

The kinetic parameters of three IRT (Inhibitor-Resistant-TEM-derived-) beta-lactamases (IRT-5, IRT-6 and IRT-I69) were determined for substrates and the beta-lactamase inhibitors: clavulanic acid, sulbactam and tazobactam, and compared with those of TEM-1 beta-lactamase. The catalytic behaviour of the beta-lactamases towards substrates and inhibitors was correlated with the properties of the amino acid at position ABL69. 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 contributing to the oxyanion hole. We suggest that hydrophobicity could be the main factor responsible for the kinetic properties of Met69Leu (IRT-5), as no steric effects could be detected by molecular modelling. Hydrophobicity and steric constraints are combined in Met69Ile and Met69Val, IRT-I69 and IRT-6, respectively.