Rate constants for a mechanism including intermediates in the interconversion of ternary complexes by horse liver alcohol dehydrogenase

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Abstract
Transient kinetic data for partial reactions of alcohol dehydrogenase and simulations of progress curves have led to estimate of rate constants for the following mechanism, at pH 8.0 and 25.degree.C: E .dblarw. E-NAD+ .dblarw. * E-NAD+ .dblarw. E-NAD+ -RCH2OH .dblarw. E-NAD+-RCH2O- .dblarw. *E-NADH-RCHO .dblarw. E-NADH-RCHO .dblarw. E-NADH .dblarw. E Previous results show that the E-NAD+ complex isomerizes with a forward rate constant of 620 s-1 [Sekhar, V.C., and Plapp, B.V. (1988) Biochemistry 27,5082-5088]. The enzyme-NAD+-alcohol complex has a pK value of 7.2 and loses a protein rapidly (> 1000 s-1). The transient oxidation of ethanol is 2-fold faster in D2O, and proton inventory results suggest that the transition state has a charge of -0.3 on the substrate oxygen. Rate constants for hydride ion transfer in the forward or reverse reactions were similar for short-chain aliphatic substrates (400-600 s-1). A small deuterium isotope effect for transient oxidation of longer chain alcohols is apparently due to the isomerization of the E-NAD+ complex. The transient reduction of aliphatic aldehydes showed no primary deuterium isotope effect; thus, an isomerization of the E-NADH-aldehyde complex is postulated, as isomerization of the E-NADH complex was too fast to be detected. The estimated microscopic rate constants show that the observed transient reactions are controlled by multiple steps.