Mutation of arginine 98, which serves as a substrate-recognition site of D-amino acid aminotransferase, can be partly compensated for by mutation of tyrosine 88 to an arginyl residue.
D-Amino acid aminotransferase is the only aminotransferase that catalyzes the transamination of D-amino acids. We studied the role of the binding site for the alpha-carboxyl group of substrates, which is presumably crucial for the unique stereospecificity of the enzyme. The site-directed mutagenesis of Arg98, which is the putative carboxyl-binding site, as judged on the basis of X-ray crystallographic studies [Sugio, S., Petsko, G.A., Manning, J.M., Soda, K., and Ringe, D. (1995) Biochemistry 34, 9661-9669], by replacement with methionine and lysine, resulted in decreases in the kmax values and increases in the Kd values for both amino donors and amino acceptors. The introduction of another mutation, that of Tyr88, which is located near Arg98 in the spacial structure, by replacement with arginine, in addition to the above Arg98 mutation, resulted in increases in the kmax values but little change in the Kd values. These results suggest that Arg98 constitutes the carboxyl-binding site for the substrate, efficient catalysis by the enzyme being facilitated upon binding. The mutant enzymes are also relieved from inhibition by high concentrations of alpha-ketoglutarate, which is an inherent character of the wild-type enzyme. Therefore, Arg98 is also responsible for the inhibition by alpha-ketoglutarate.
Kishimoto
K
K
Institute for Chemical Research, Kyoto University, Uji.
Yoshimura
T
T
Soda
K
K
Esaki
N
N
D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids / Q000494:pharmacology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D013379:Substrate Specificity; D000637:Transaminases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D007656:Ketoglutaric Acids; D014443:Tyrosine; D001120:Arginine; D000637:Transaminases