A reinvestigation of the single-turnover time course for the horse liver alcohol dehydrogenase (LADH) catalyzed oxidation of NADH and of (4R)-4-deuterio-NADH (NADD) by a series of substituted benzaldehyde substrates has been carried out in the alkaline pH range. The reaction time courses were monitored at 330 nm via stopped-flow rapid-mixing spectrophotometry. The pyrazole “suicide technique” [McFarland, J. T., & Bernhard, S. A. (1972) Biochemistry 11, 1486-1493] has been employed to limit reaction to a single turnover when enzyme sites are limiting. In all cases, the time courses were found to be remarkably biphasic under conditions of substrate and NADH (or NADD) saturation. As reported previously (McFarland & Bernhard, 1972), the two amplitudes of reaction at substrate saturation are not necessarily equal, but the ratio of amplitudes is not far from unity (5 > Afast/Ashow >0.8). On-line computer analysis demonstrated that the time courses were best described by the rate law for two simultaneous first-order processes [OD = OD„ + B expC-k1t) + Cexp(-k2t)]- Amplitude analysis [involving corrections for instrument dead time and for the OD changes contributed to the slow step by the formation of the E(NAD-pyrazole)ad-duct] gave the following percentage amplitudes for the rapid phase of the transient time course: p-CH3O-, 62 ± 10%; p-CH3-, 70± 10%; p-H-, 65± 10%; p-Cl-, 66± 10%; and p-NO2-, 50 ± 10%. Control experiments in which the concentration of NADH rather than enzyme sites was limiting demonstrate that the rates and amplitudes which characterize the biphasic time course are unaffected by this change. Likewise, the presence of 0.44 mM NAD+ in the stopped-flow syringe containing pyrazole and a saturating concentration of substrate was found to exert no influence on the biphasic time courses. The relative insensitivity of the ratio of amplitudes in the two phases to wide variations in the electronic substituents on the substituted benzaldehyde substrates argues strongly against a compulsory ordered mechanism as an explanation of kinetic biphasicity. The insensitivity of the amplitude ratio to the (rate-dependent) distinction between NADH and NADD adds further weight to the argument that the compulsory-ordered mechanism (although correct) is insufficient to account for kinetic biphasicity. © 1979, American Chemical Society. All rights reserved.
