THE C-TERMINAL LOOP OF ALDEHYDE REDUCTASE DETERMINES THE SUBSTRATE AND INHIBITOR SPECIFICITY

Human aldehyde reductase has a preference for carboxyl group-containin g negatively charged substrates. It belongs to the NADPH-dependent ald o-keto reductase superfamily whose members are in part distinguished b y unique C-terminal loops. To probe the role of the C-terminal loops i n determining substrate specificities in these enzymes, two arginine r esidues, Arg308 and Arg311, located in the C-terminal loop of aldehyde reductase, and not found in any other C-terminal loop, were replaced with alanine residues. The catalytic efficiency of the R311A mutant fo r aldehydes containing a carboxyl group is reduced 150-250-fold in com parison to that of the wild-type enzyme, while substrates not containi ng a negative charge are unaffected. The R311A mutant is also signific antly less sensitive to inhibition by dicarboxylic acids, indicating t hat Arg311 interacts with one of the carboxyl groups. The inhibition p attern indicates that the other carboxyl group binds to the anion bind ing site formed by Tyr49, His112, and the nicotinamide moiety of NADP( +). The correlation between inhibitor potency and the length of the di carboxylic acid molecules suggests a distance of approximately 10 Angs trom between the amino group of Arg311 and the anion binding site in t he aldehyde reductase molecule. The sensitivity of inhibition of the R 311A mutant by several commercially available aldose reductase inhibit ors (ARIs) was variable, with tolrestat and zopolrestat becoming more potent inhibitors (30- and 5-fold, respectively), while others remaine d the same or became less potent, The catalytic properties, substrate specificity, and susceptibility to inhibition of the R308A mutant rema ined similar to that of the wild-type enzyme. The data provide direct evidence for C-terminal loop participation in determining substrate an d inhibitor specificity of aldo-keto reductases and specifically ident ifies Arg311 as the basis for the carboxyl-containing substrate prefer ence of aldehyde reductase.