Enzyme kinetic studies with beta-lactamases against cefotaxime and other cephalosporins
Yan, S.Y.; Li, J.T.
Chinese Medical Journal 100(12): 940-944
1987
ISSN/ISBN: 0366-6999 PMID: 3133166 Document Number: 286619
Enzyme stability and enzyme kinetic studies with plasmid-mediated and chromosome-mediated beta-lactamases against cefotaxime, cefoperazone and other cephalosporins were performed using spectrophotometry. Beta-lactamases were extracted from gram-negative organisms and identified by isoelectric focusing with the special reagent nitrocefin. The relative hydrolysis rate (%), Michaelis-Menten constant (Km), maximum velocity (Vmax) and inhibition constant (Ki) were obtained and used as indicators for comparative study of enzyme stability in six cephalosporins. The affinity of compounds binding to target sites of beta-lactamases was also determined. The results show that cefotaxime is the most enzyme stable drug among the cephalosporins studied. The Vmax of TEM-1, SHV-1 and P99 enzyme hydrolyzing cefotaxime was less than 0.10% as compared with cephaloridine. Cefotaxime demonstrates high affinity to the chromosome-mediated enzyme P99, and its Ki value is 0.035 .mu.m. Cefoperazone exhibits less enzyme inhibitory activity and is unstable with most of the beta-lactamases used.