Probing the active site of human aldose reductase. Site-directed mutagenesis of Asp-43, Tyr-48, Lys-77, and His-110.

摘要: Structural models of human aldose reductase complexed with NADPH have revealed the apposition C4 nicotinamide ring tyrosine 48 and histidine 110, suggesting that either these residues could function as proton donor in reaction mechanism. Tyrosine is also part a hydrogen-bonding network includes lysine 77 aspartate 43. In order to study potential catalytic roles 4 residues, we evaluated kinetic properties mutants containing structurally conservative replacements at sites. Enzymatic activity was undetectable when Tyr-48 mutated phenylalanine (Y48F) although affinity for unchanged. contrast, mutant asparagine substituted His-110 (H110N) characterized by an almost 80,000-fold increase Km, but only about 14-fold reduction kcat measured D-glyceraldehyde. Modest changes were observed 43 (D43N): Km aldehyde substrates elevated up 17-fold, decreased less than 16-fold. However, Kd(NADP) values D43N 5 times higher those wild type. Mutant enzyme methionine (K77M) 1,460-fold active These results are consistent acting acid-base catalyst confirm importance Asp-43, Lys-77, structure site.