Biochemical characteristics of extended broad spectrum beta-lactamases

Extended broad spectrum beta-lactamases such as TEM-3 (CTX-1), TEM-5 (CAZ-1), TEM-10 and RHH-1 were purified and found to have lower specific activities than the TEM-1 or TEM-2 beta-lactamases. Total hydrolytic activity in crude extracts was also lower for the extended broad spectrum enzymes. These beta-lactamases hydrolyzed not only penicillins such as carbenicillin, cloxacillin and piperacillin, but also cephalosporins and monobactams. The most notable differences in substrate profiles between the extended broad spectrum enzymes and TEM-2 enzymes occurred with oxime-containing antibiotics. Although all the extended broad spectrum enzymes described above hydrolyzed cefotaxime, ceftazidime and aztreonam, the four enzymes could be easily differentiated: TEM-3 hydrolyzed cefotaxime preferentially, TEM-5 and RHH-1 hydrolyzed ceftazidime approximately three times faster than cefotaxime, whereas TEM-10 hydrolyzed ceftazidime 42 times faster than cefotaxime. All the enzymes were inhibited well by clavulanic acid, with I50 values ranging from 4.3 to 12 nM, compared to 130 nM for TEM-2. Inhibition by sulbactam was also better for the extended broad spectrum than for the TEM-2 beta-lactamases, with I50 values of 12-940 nM for the extended broad spectrum enzymes, compared to 1600 nM for the TEM-2 beta-lactamase.