Mechanistic basis for nonlinear kinetics of aldehyde reduction catalyzed by aldose reductase

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Abstract
Bovine kidney aldose reductase (ALR2) displays substrate inhibition by aldehyde substrates that is uncompetitive versus NADPH when allowance is made for nonenzymatic reaction of the aldehyde with the adenine moiety of NADPH. A time-dependent increase in substrate inhibition observed in product versus time plots for reduction of short-chain aldoses containing an enolizable .alpha.-proton, but not for p-nitrobenzaldehyde, is shown to be consistent with a model in which rapidly reversible inhibition due to formation of the dead-end E .cntdot. NADP .cntdot. glycolaldehyde complex is combined with the formation at the enzyme active site of a tightly-bound covalent NADP-glycolaldehyde adduct. Quantitative analysis of reaction time courses for ALR2-catalyzed reduction of glycoaldehyde using NADPH or the 3-acetylpyridine analogue, (AP)ADPH, yields values of the forward and reverse rate constants for ALR2-mediated adduct formation that agree with the values determined in the absence of glycoladehyde turnover. Substrate inhibition is only partial, indicating that reaction can occur via an alternate pathway at high [glycolaldehyde]. Kinetic evidence for a slow isomerization of the E .cntdot. NADP complex at pH 8.0 is used to explain the similar V/Et values observed for glycolaldehyde reduction at pH 7.0 using NAPDH, (AP)ADPH, and the hypoxanthine analogue N(Hx)DPH. The practical implicatiions of these results for kinetic studies of aldose reductase are discussed.

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