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

Abstract

A method is reported for automating measurements of K_m and specific V_max values from single progress curves of hydrolysis of β-lactam antibiotics by crude (or purified) β-lactamase preparations. The method is based on the half-time analysis procedure of Wharton & Szawelski (1982). The specific V_max 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 K_m and specific V_max values, rather than relative rates of hydrolysis, in surveys of bacterial β-lactamases.