Rapid and automated measurement of Km and specific Vmax values of beta-lactamases in bacterial extracts

A method is reported for automating measurements of Km and specific Vmax values from single progress curves of hydrolysis of beta-lactam antibiotics by crude (or purified) beta-lactamase preparations. The method is based on the half-time analysis procedure of Wharton & Szawelski (1982). The specific Vmax is obtained in units of mols of substrate hydrolysed /min/mg dry wt of cells, but could be expressed as easily in terms of mols of enzyme or mg of protein. We propose that the method will allow reporting of Km and specific Vmax values, rather than relative rates of hydrolysis, in surveys of bacterial beta-lactamases.