Site-directed mutagenesis of His-42, His-188 and Lys-263 of human aldose reductase.
The role of His42, His188, and Lys263 residues in the catalytic action of human aldose reductase was investigated in association with various inhibitors of this enzyme by site-directed mutagenesis. While mutations at His42- greater than Gln, His42- greater than Tyr, His188- greater than Gln, and His188- greater than Tyr brought small change in the kinetic parameters, Lys263- greater than Glu mutation markedly increased the Km value for the substrate DL-glyceraldehyde by a factor of 60. Lys263- greater than Met substitution resulted in approximately 14 fold elevation of Km for the substrate. By contrast, mutation of Lys263- greater than Arg significantly decreased the Km for the substrate with concomitant reduction in kcat. Moderate increase in Km values for the cofactor NADPH was demonstrated for mutated enzymes. These results are indicative of the possible role of Lys263 in the substrate binding through electrostatic interaction. The inhibitor constants (Ki) for structurally diverse aldose reductase inhibitors against mutated enzymes demonstrated different degree of alteration, indicating binding sites of aldose reductase inhibitors on the enzyme molecule vary from one another, and some of the sites are more closely correlated with the physicochemical property of Lys263.
Yamaoka
T
T
Department of Pediatric Pharmacology, National Children's Medical Research Center, Tokyo, Japan.
Matsuura
Y
Y
Yamashita
K
K
Tanimoto
T
T
Nishimura
C
C
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D005449:Fluorenes / Q000494:pharmacology; D006801:Humans; D006827:Hydantoins / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D048289:Imidazolidines; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D013329:Structure-Activity Relationship
D005449:Fluorenes; D006827:Hydantoins; D007093:Imidazoles; D048289:Imidazolidines; D009281:Naphthalenes; C052241:imirestat; C040550:tolrestat; D009249:NADP; D000449:Aldehyde Reductase; C026411:sorbinil