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Investigates the role of asparagine 152 in the catalytic hydrolysis of beta-lactam by AmpC beta-lactamase of Escherichia coli. Effect of site-directed mutagenesis on the activity of AmpC beta-lactamase toward substrates and inhibitors; Comparison with other mutated classes of penicillin-sensitive enzymes.

Examines the kinetics of beta-lactam hydrolysis by wild-type AmpC beta-lactamase from Escherichia coli and three mutant proteins created by substitution of tyrosine 150. Construction of plasmids and mutagenesis; Physical properties of mutants; Effects of mutations on kinetic properties.

Examines the role of the highly conserved lysine315 residue in the catalytic mechanism of a class C beta-lactamase. Use of site-directed mutagenesis; Effects of the mutations on the kinetics of penicillin G and cephalothin turnover; Pre-steady-state kinetics with carbenicillin at different pH; Activity at acidic pH.

By challenging the efficiency of some of our most useful antimicrobial weapons, bacterial antibiotic resistance is becoming an increasingly worrying clinical problem. A good antibiotic is expected to exhibit a high affinity for its target and to reach it rapidly, while escaping chemical modification by inactivating enzymes and elimination by efflux mechanisms. A study of the behaviour of a β-lactamase-overproducing mutant of Enterobacter cloacae in the presence of several penicillins and cephalosporins showed that the minimum inhibitory concentration (MIC) values for several compounds were practically independent of the sensitivity of the target penicillin binding protein (PBP), even for poor β-lactamase substrates. This apparent paradox was explained by analysing the equation that relates the antibiotic concentration in the periplasm to that in the external medium. Indeed, under conditions that are encountered frequently in clinical isolates, the factor characterizing the PBP sensitivity became negligible. The conclusions can be extended to all antibiotics that are sensitive to enzymatic inactivation and efflux mechanisms and must overcome permeability barriers. It would be a grave mistake to neglect these considerations in the design of future antibacterial chemotherapeutic agents. [ABSTRACT FROM AUTHOR]