Novel methods to estimate the enantiomeric ratio and the kinetic parameters of enantiospecific enzymatic reactions

The Enantiomeric Ratio (E) of the enzyme, acting as specific catalysts in resolution of enantiomers, is an important parameter in the quantitative description of these chiral resolution processes. In the present work, two novel methods hereby called Method I and II, for estimating E and the kinetic parameters Km and Vm of enantiomers were developed. These methods are based upon initial rate (v) measurements using different concentrations of enantiomeric mixtures (C) with several molar fractions of the substrate (x). Both methods were tested using simulated “experimental data” and actual experimental data. Method I is easier to use than Method II but requires that one of the enantiomers is available in pure form. Method II, besides not requiring the enantiomers in pure form shown better results, as indicated by the magnitude of the standard errors of estimates. The theoretical predictions were experimentally confirmed by using the oxidation of 2-butanol and 2-pentanol catalyzed by Thermoanaerobium brockii alcohol dehydrogenase as reaction models. The parameters E, Km and Vm were estimated by Methods I and II with precision and were not significantly different from those obtained experimentally by direct estimation of E from the kinetic parameters of each enantiomer available in pure form.