MECHANISM OF HUMAN ALDEHYDE REDUCTASE - CHARACTERIZATION OF THE ACTIVE-SITE POCKET

Human aldehyde reductase is a NADPH-dependent aldo-keto reductase that is closely related (65% identity) to aldose reductase, an enzyme invo lved in the pathogenesis of some diabetic and galactosemic complicatio ns. Ln aldose reductase, the active site residue Tyr48 is the proton d onor in a hydrogen-bonding network involving residues Asp43/Lys77, whi le His110 directs the orientation of substrates in the active site poc ket. Mutation of the homologous Tyr49 to phenylalanine or histidine (Y 49F or Y49H) and of Lys79 to methionine (K79M) in aldehyde reductase y ields inactive enzymes, indicating similar roles for these residues in the catalytic mechanism of aldehyde reductase. A H112Q mutant aldehyd e reductase exhibited a substantial decrease in catalytic efficiency ( k(cat)/K-m) for hydrophilic (average 150-fold) and aromatic substrates (average 4200-fold) and 50-fold higher IC50 values for a variety of i nhibitors than that of the wild-type enzyme. The data suggest that His 112 plays a major role in determining the substrate specificity of ald ehyde reductase, similar to that shown earlier for the homologous His1 10 in aldose reductase [Bohren, K. M., et. al, (1994) Biochemistry 33, 2021-2032]. Mutation of Ile298 or Val299 affected the kinetic paramet ers to a much lesser degree. Unlike native aldose reductase, which con tains a thiol-sensitive Cys298, neither the I298C or V299C mutant exhi bited any thiol sensitivity, suggesting a geometry of the active site pocket different from that in aldose reductase. Also different from al dose reductase, the detection of a significant primary deuterium isoto pe effect on k(cat) (1.48 +/- 0.02) shows that nucleotide exchange is only partially rate-limiting. Primary substrate and solvent deuterium isotope effects on the H112Q mutant suggest that hydride and proton tr ansfers occur in two discrete steps with hydride transfer taking place first. Dissociation constants and spectroscopic and fluorimetric prop erties of nucleotide complexes with various mutants suggest that, in a ddition to Tyr49 and His112, Lys79 plays a hitherto unappreciated role in nucleotide binding. The mode of inhibition of aldehyde reductase b y aldose reductase inhibitors (ARIs) is generally similar to that of a ldose reductase and involves binding to the E:NADP(+) complex, as show n by kinetic and direct inhibitor-binding experiments, The order of AR I potency was AL1576 (K-i = 60 nM) > tolrestat > ponalrestat > sorbini l > FK366 > zopolrestat > alrestatin (K-i = 148 mu M). Our data on ald ehyde reductase suggest that the active site pocket significantly diff ers from that of aldose reductase, possibly due to the participation o f the C-terminal loop in its formation.