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 48 and histidine 110, suggesting that either of these residues could function as the proton donor in the reaction mechanism. Tyrosine 48 is also part of a hydrogen-bonding network that includes lysine 77 and aspartate 43. In order to study the potential catalytic roles of these 4 residues, we evaluated the kinetic properties of mutants containing structurally conservative replacements at these sites. Enzymatic activity was undetectable when Tyr-48 was mutated to phenylalanine (Y48F) although affinity for NADPH was unchanged. In contrast, a mutant containing asparagine substituted for His-110 (H110N) was characterized by an almost 80,000-fold increase in Km, but only about a 14-fold reduction in kcat measured with D-glyceraldehyde. Modest changes in catalytic properties were observed in the mutant containing aspartate 43 substituted with asparagine (D43N): Km for aldehyde substrates was elevated up to 17-fold, and kcat decreased less than 16-fold. However, the Kd(NADP) values for D43N were about 5 times higher than those for wild type. Mutant enzyme containing methionine substituted for lysine 77 (K77M) was up to 1,460-fold less active than the wild type. These results are consistent with Tyr-48 acting as the acid-base catalyst in human aldose reductase and confirm the importance of Asp-43, Lys-77, and His-110 to the structure and function of the active site.
Tarle
I
I
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110.
Borhani
D W
DW
Wilson
D K
DK
Quiocho
F A
FA
Petrash
J M
JM
D000449:Aldehyde Reductase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D005260:Female; D006639:Histidine; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014443:Tyrosine
D017931:DNA Primers; D011994:Recombinant Proteins; D001224:Aspartic Acid; D014443:Tyrosine; D006639:Histidine; D009249:NADP; D000449:Aldehyde Reductase; D008239:Lysine