TYROSINE-48 IS THE PROTON DONOR AND HISTIDINE-110 DIRECTS SUBSTRATE STEREOCHEMICAL SELECTIVITY IN THE REDUCTION REACTION OF HUMAN ALDOSE REDUCTASE - ENZYME-KINETICS AND CRYSTAL-STRUCTURE OF THE Y48H MUTANT ENZYME

The active site of human aldose reductase contains two residues, His11 0 and Tyr48, either of which could be the proton donor during catalysi s. Tyr48 is a candidate since its hydroxyl group is in proximity to Ly s77 and thus may have an abnormally low pK(a) value. To distinguish be tween these possibilities, we used site-directed mutagenesis to create the H110Q and H110A, the Y48F, Y48H, and Y48S, and the K77M mutant en zymes. The two His110 mutants resulted in a 1000-20 000-fold drop in k (cat)/K-m, respectively, for the reduction of DL-glyceraldehyde at pH 7. The Y48F mutation caused total loss of activity, whereas the Y48H a nd Y48S mutants retained catalytic activity with k(cat)/K-m reduced by 5 orders of magnitude. The K77M mutant is an inactive enzyme. Kinetic studies using xylose stereoisomers show that the wildtype enzyme dist inguishes between D-xylose, L-xylose, and D-lyxose up to 150-fold bett er than the H110A or H110Q mutants. The His110 mutants do not effectiv ely discriminate between these isomers (4-11-fold). The crystal struct ure of the Y48H mutant refined at 1.8-Angstrom resolution shows that t he overall structure is not significantly different from the wild-type structure. Electron densities for the histidine side chain and a new water molecule fill the space occupied by Tyr48 in the wild-type enzym e. The water molecule is in hydrogen-bonding distance to the N zeta gr oup of Lys77 and to the N epsilon Of His48 and fills the space occupie d by the hydroxyl group of tyrosine in the wild-type structure. These findings suggest that proton transfer is mediated in the Y48H mutant e nzyme by the water molecule. The Y48H mutant shows large and equal pri mary deuterium isotope effects on k(cat) and k(cat)/K-m (1.81 +/- 0.03 ), providing direct evidence for hydride transfer as the rate-determin ing step in this mutant. Deuterium solvent isotope effects indicate th at the relative contribution of proton transfer to this step of the ca talytic cascade is much less important for the Y48H mutant than for th e wild-type enzyme [(D2O)(k(cat)/K-m) = 1.06 +/- 0.02 and 4.73 +/- 0.2 3, respectively]. The kinetic and mutagenesis data, together with stru ctural data, indicate that His110 plays an important role in the orien tation of substrates in the active site pocket, while Tyr48 is the pro ton donor during aldehyde reduction by aldose reductase.