MASS-SPECTRAL KINETIC-STUDY OF ACYLATION AND DEACYLATION DURING THE HYDROLYSIS OF PENICILLINS AND CEFOTAXIME BY BETA-LACTAMASE TEM-1 AND THE G238S MUTANT

The G238S substitution found in extended-spectrum natural mutants of T EM-1 beta-lactamase induces a new capacity to hydrolyze cefotaxime and a large loss of activity against the good substrates of TEM-1. To und erstand this phenomenon at the molecular level, a method to determine the acylation and deacylation elementary rate constants has been devel oped by using electrospray mass spectrometry combined with UV spectrop hotometry. The hydrolysis of penicillins and cefotaxime by TEM-1 and t he G238S mutant shows that the behavior of penicillins and cefotaxime is very different. With both enzymes, the limiting step is deacylation for penicillin hydrolysis, but acylation for cefotaxime hydrolysis. F urther analyses of the G238S mutant show that the loss of activity aga inst penicillins is due to a large decrease in the deacylation rate an d that the increase in catalytic efficiency against cefotaxime is the result of a better K-m and an increased acylation rate. These modifica tions of the elementary rate constants and the hydrolytic capacity in the G238S mutant could be linked to structural effects on the Omega-lo op conformation in the active site.