THE ASPARAGINE TO ASPARTIC-ACID SUBSTITUTION AT POSITION-276 OF TEM-35 AND TEM-36 IS INVOLVED IN THE BETA-LACTAMASE RESISTANCE TO CLAVULANIC ACID

TEM-35 (inhibitor resistant TEM (IRT)-4) and TEM-36 (IRT-7) clavulanic acid-resistant beta-lactamases have evolved from TEM-1 beta-lactamase by two substitutions: a methionine to a leucine or a valine at position 69 and an asparagine to an aspartic acid at position 276. The substitutions at position 69 have previously been shown to be responsible for the resistance to clavulanic acid, and they are the only mutations encountered in TEM-33 (IRT-5) and TEM-34 (IRT-6). However, the N276D substitution has never been found alone in inhibitor-resistant beta-lactamases, and its role in resistance to clavulanic acid was thus unclear, The N276D mutant was constructed, purified, and kinetically characterized, It was shown that the substitution has a direct effect on substrate affinities and leads to slightly decreased catalytic efficiencies and that clavulanic acid becomes a poor substrate of the enzyme, Electrospray mass spectrometry demonstrated the simultaneous presence of free and inhibited enzymes after incubation with clavulanic acid and showed that a cleaved moiety of clavulanic acid leads to the formation of the major inactive complex, The kinetic properties of the N276D mutant could be linked to a salt-bridge interaction of aspartic acid 276 with arginine 244 that alters the electrostatic properties in the substrate binding area.