ENZYMATIC ASSAY OF BETA-LACTAMASE USING CIRCULAR-DICHROISM SPECTROPOLARIMETRY

A method for measuring the rates of enzymatic hydrolysis of beta-lacta m antibiotics based on circular dichroism spectropolarimetry is descri bed. Unhydrolyzed beta-lactam antibiotics have high molar ellipticitie s, but the hydrolyzed compounds are circular dichroism (CD) inactive i n the case of penams or have significantly different CD spectra in the case of cephems. By measuring CD at constant wavelength as a function of time for reaction mixtures containing beta-lactamase and beta-lact am antibiotics, rates of hydrolysis and steady-state enzyme kinetic co nstants can be derived. The method was applied to measurement of a wid e range of enzymatic reaction constants for wild-type and four mutant RTEM-1 beta-lactamases. Compared to the commonly employed assay based on ultraviolet spectroscopy, the new method offers several advantages. These include the ability to measure larger enzymatic Michaelis-Mente n constants, less interference from high concentrations of beta-lactam ase, higher sensitivity, and potentially less interference from other uv-absorbing components of complex reaction mixtures. (C) 1997 Academi c Press.