CRITICAL HYDROGEN-BONDING BY SERINE 235 FOR CEPHALOSPORINASE ACTIVITYOF TEM-1 BETA-LACTAMASE

The role of Ser-235 in the catalytic mechanism of the TEM-1 beta-lacta mase has been explored by the study of a mutant enzyme in which Ser-23 5 has been substituted by alanine (Ala-235 mutant enzyme). A comparati ve kinetic analysis of both the wild-type and the Ala-235 TEM-1 enzyme s revealed little effect of this substitution of residue 235 on the tu rnover of penicillins but a greater effect on the turnover of cephalos porins. Susceptibility testing of Escherichia coli strains harboring t he wild-type TEM-1 beta-lactamase and the Ala-235 mutant enzyme reveal ed an effect of the mutation similar to that observed in the enzymolog ical studies. The MICs of two representative cephalosporins for the st rain containing the mutant enzyme were much lower than those for the i sogenic strain bearing the wild-type TEM-1 beta-lactamase. On the othe r hand, the strain with the mutant enzyme was still highly resistant t o penicillins.