Evidence that the slow conformation change controlling NADH release from the enzyme is rate-limiting during the oxidation of propionaldehyde by aldehyde dehydrogenase

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Abstract
The displacement of NADH from the aldehyde dehydrogenase .cntdot. NADH complex by NAD+ was followed at pH 7.0, and the data were fitted by a non-linear least-squares iterative procedure. At pH 7.0 the decay constants for the dissociation of NADH from aldehyde dehydrogenase .cntdot. NADH complexes (1.62 .+-. 0.09 s-1 and 0.25 .+-. 0.004 s-1) were similar to the values previously determined by MacGibbon, Buckley and Blackwell [(1977) Biochem. J. 165, 455-462] at pH 7.6, and apparent differences between these values and those reported by Dickinson [(1985) Biochem. J. 225, 159-165] are resolved. Experiments at low concentrations of propionaldehyde show that isomerization of a binary E* .cntdot. NADH complex is part of the normal catalytic mechanism of the enzyme. Evidence is presented that the active-site concentration of aldehyde dehydrogenase is halved when enzyme is pre-diluted to low concentrations before addition of NAD+ and substrate. The consequences of this for the reported values of kcat. are discussed. A general mechanism for the aldehyde dehydrogenase-catalysed oxidation of propionaldehyde which accounts for the published kinetic data, at concentrations of aldehyde which bind only at the active site, is presented.