Further investigations of the transient kinetics of alcohol oxidation catalysed by horse liver alcohol dehydrogenase

Abstract

Due to the controversy over the half-of-the-sites reactivity of horse liver alcohol dehydrogenase during benzyl alcohol oxidation, we have re-investigated the transient kinetics, stoichiometry and rate parameters over a wide range of substrate concentrations (0.05 m m to 40 m m) at pH 7.0 and 8.5 and using newly determined extinction coefficients. Data were elaborated by computer analysis in order to separate the initial rapid step (burst) from the whole time-course of the reaction. It has been found that: (1) the dependence of the burst amplitude upon benzyl alcohol concentration is distinctly biphasic. In the range from 0.05 m m up to approximately 1 m m the burst amplitude is rather insensitive to changes in alcohol concentration and corresponds to 50% of the active sites of the enzyme; for alcohol concentrations greater than 1 m m this amplitude increases and reaches a value of approximately 90% when benzyl alcohol is 40 m m. (2) The steady-state initial rate is also biphasic with respect to alcohol concentration, indicative of substrate inhibition, which begins in the concentration range at which deviation from the half-burst also appears. In other words, burst amplitudes larger than 50% are concomitant with inhibition of the rate of enzyme turnover. (3) In the presence of isobutyramide the burst is larger than 50% for the whole range of concentration of the substrate and extrapolates at infinite substrate concentration to approximately 90% of the enzyme sites. (4) With deuteroethanol as substrate, the burst is larger than 50%, with or without isobutyramide, and extrapolates to approximately 95% of the enzyme sites at infinite substrate concentration. These data explain the discrepancy of results in the literature concerning the transient kinetics of alcohol oxidation. Mechanistic implications of the results (particularly the deviation from the halfof-the-sites behaviour of benzyl alcohol under inhibition conditions) are discussed.