Mechanistic studies on equine liver alcohol dehydrogenase. I. Stoichiometry relation of the coenzyme binding sites to the catalytic sites active in the reduction of aromatic aldehydes in the transient state

Abstract

The course of the transient kinetics for the equine liver alcohol dehydrogenase catalyzed reduction of chromophoric aromatic aldehydes by reduced nicotinamide-adenine dinucleotide has been studied in the pH region 8-10 using rapid-mixing stopped-flow spectrophotometric instrumentation. Two kinetic processes, well separated in rate, are observed for the conversion of reactants into products under conditions of excess enzyme. The amplitude of the opticaldensity change accompanying the rapid initial step corresponds to the conversion of exactly one-half of the limiting substrate (aldehyde or reduced nicotinamide-adenine dinucleotide) to product (alcohol or oxidized nicotinamide-adenine dinucleotide). The slower second process accounts for the conversion of the remaining material into product and is found to have a first-order rate numerically similar to the steady-state turnover number. When reduced nicotinamideadenine dinucleotide and aldehyde are present in excess of enzyme, there occurs a rapid initial process, prior to the attainment of the steady state, with an optical-density change corresponding to the conversion of substrates (aldehyde T J.he nicotinamide-adenine dinucleotide requiring enzyme horse liver alcohol dehydrogenase (EC 1.1.1.1) catalyzes the interconversion of aldehydes and alcohols (eq 1) with equilibrium thermodynamically favored in the direction shown for most substrates at accessible pH (Sund and Theorell, 1962).

Kinetic Transients in the Reduction of Aldehydes Catalysed by Liver Alcohol Dehydrogenase

“…As reported previously [11,16] and illustrated by Fig. S, the biphasic kinetic pattern persists under such conditions, and amplitudes and time constants for the two transients were determined at a fixed high concentration of NADH for a series of enzyme concentrations intermediate between the concentrations of substrate and coenzyme.…”

Approaches to the study of enzyme mechanisms lactate dehydrogenase

“…3 and 4). Since there are several reports in the literature [74] which are in convlict with our conclusion that the three enzymes discussed here have thermodynamically and kinetically independent sites, some effects which give apparent non equivalence must be discussed. The transient or single turnover can be described as the sum of a series of exponentials.…”

Effect of pH on the Binding of Decanoate and Trifluoroethanol to Liver Alcohol Dehydrogenase

“…Crystalline horse-liver alcohol dehydrogenase from Boehringer Mannheim GmbH was further purified as described by Bernhard et al [3]. When required, unbuffered enzyme solutions were prepared using 33 mM sodium sulphate for elution of the enzyme in the column chromatographic purification step.…”