Abstract
A method for measuring the rates of enzymatic hydrolysis of β-lactam antibiotics based on circular dichroism spectropolarimetry is described. Unhydrolyzed β-lactam antibiotics have high molar ellipticities, but the hydrolyzed compounds are circular dichroism (CD) inactive in 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 β-lactamase and β-lactam antibiotics, rates of hydrolysis and steady-state enzyme kinetic constants can be derived. The method was applied to measurement of a wide range of enzymatic reaction constants for wild-type and four mutant RTEM-1 β-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–Menten constants, less interference from high concentrations of β-lactamase, higher sensitivity, and potentially less interference from other uv-absorbing components of complex reaction mixtures.
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