Mono- and binuclear Zn2+-beta-lactamase - Role of the conserved cysteine in the catalytic mechanism

When expressed by pathogenic bacteria, Zn2+-beta-lactamases induce resistan ce to most beta-lactam antibiotics. A possible strategy to fight these bact eria would be a combined therapy with non-toxic inhibitors of Zn2+-beta-lac tamases together with standard antibiotics. For this purpose, it is importa nt to verify that the inhibitor is effective under all clinical conditions. We have investigated the correlation between the number of zinc ions bound to the Zn2+-beta-lactamase from Bacillus cereus and hydrolysis of benzylpe nicillin and nitrocefin for the wild type and a mutant where cysteine 168 i s replaced by alanine. It is shown that both the mono-Zn2+ (mononuclear) an d di-Zn2+ (binuclear) Zn2+-beta-lactamases are catalytically active but wit h different kinetic properties, The mono-Zn2+-beta-lactamase requires the c onserved cysteine residue for hydrolysis of the beta-lactam ring in contras t to the binuclear enzyme where the cysteine residue is not essential. Subs trate affinity is not significantly affected by the mutation for the mononu clear enzyme but is decreased for the binuclear enzyme. These results were derived from kinetic studies on two wild types and the mutant enzyme with b enzylpenicillin and nitrocefin as substrates. Thus, targeting drug design t o modify this residue might represent an efficient strategy, the more so if it also interferes with the formation of the binuclear enzyme.