Site-directed mutagenesis at the active site of Escherichia coli TEM-1 beta-lactamase. Suicide inhibitor-resistant mutants reveal the role of arginine 244 and methionine 69 in catalysis.

Arginine 244 is a highly conserved residue in Class A beta-lactamases, while methionine 69 is not. Informational suppression experiments show that replacement of M69 by a leucine, or that of R244 by most other amino acids lead to clavulanic acid-resistant phenotypes. The arginyl 244 side chain is tightly held in a network of interactions within the active site. Its replacement by a glutamine or a threonine perturbs the enzyme kinetics but to a smaller extent than would have been predicted if it were directly involved in substrate binding. Clavulanic acid and sulbactam still interact specifically with the mutant enzymes but are much less efficiently metabolized. Substitutions at position 244 also unveil interactions between the C6 substituent of substrates and the Asn132/Glu104 region of the active site. Methionine 69 is located in a region of strong structural constraints and presents an unusual conformation. Molecular dynamics simulation showed that its replacement by a leucine does not release the strain in this area and induces only minor structural changes. Accordingly, the kinetic behavior of the mutant is only marginally perturbed, except for suicide inhibitors. Both clavulanic acid and sulbactam are well degraded by the mutant enzyme, while irreversible inactivation is dramatically decreased. The contribution of both residues to catalysis is discussed in the light of the kinetic and structural data.