PROBING THE ACTIVE-SITE OF HUMAN ALDOSE REDUCTASE - SITE-DIRECTED MUTAGENESIS OF ASP-43, TYR-48, LYS-77, AND HIS-110

Structural models of human aldose reductase complexed with NADPH have revealed the apposition of C4 of the nicotinamide ring with tyrosine 4 8 and histidine 110, suggesting that either of these residues could fu nction as the proton donor in the reaction mechanism. Tyrosine 48 is a lso part of a hydrogen-bonding network that includes lysine 77 and asp artate 43. In order to study the potential catalytic roles of these 4 residues, we evaluated the kinetic properties of mutants containing st ructurally conservative replacements at these sites. Enzymatic activit y was undetectable when Tyr-48 was mutated to phenylalanine (Y48F) alt hough affinity for NADPH was unchanged. In contrast, a mutant containi ng asparagine substituted for His-110 (H110N) was characterized by an almost 80,000-fold increase in K(m), but only about a 14-fold reductio n in k(cat) measured with D-glyceraldehyde. Modest changes in catalyti c properties were observed in the mutant containing aspartate 43 subst ituted with asparagine (D43N): K(m) for aldehyde substrates was elevat ed up to 17-fold, and k(cat) decreased less than 16-fold. However, the K(d(NADP)) values for D43N were about 5 times higher than those for w ild type. Mutant enzyme containing methionine substituted for lysine 7 7 (K77M) was up to 1,460-fold less active than the wild type. These re sults are consistent with Tyr-48 acting as the acid-base catalyst in h uman aldose reductase and confirm the importance of Asp-43, Lys-77, an d His-110 to the structure and function of the active site.