The Significance of Amino Acid Residue Asp446 for Enzymatic Stability of Rat UDP-Glucuronosyltransferase UGT1A6

Asp446 in rat UDP-glucuronosyltransferase (UGT), UGT1A6, is an essential amino acid residue for its enzymatic activity (H. Iwanoet al. Biochem. J.325, 587–591, 1997). The role of Asp446 in UGT1A6 was investigated by comparing some properties of UGT mutant proteins that have a single mutation (D446K, D446E, D446N, D446Q, D446A, and D446T). These mutants, except D446K, had catalytic activities toward 1-naphthol and 4-methylumbelliferone. The UGT activities of D446E and D446N were about half of that of the wild type, and the activities of the other mutants were only about 1/5–1/10 of that of the wild type. TheKmvalues for 1-naphthol of these mutants were similar to that of the wild type, while the values for UDP-glucuronic acid were slightly higher. The mutants were unstable in a low-pH buffer solution and were dramatically inactivated by heat treatments. Interestingly, after 30 min of treatment at 37°C in the presence of UDP-glucuronic acid, the UGT activities of all functional mutants were elevated. These results suggest that Asp446 is an indispensable residue for folding a functional conformation of rat UGT1A6 by cooperation with UDP-glucuronic acid.