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. Iwano et 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. The Km values 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 degrees 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.
School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, 069-0836, Japan.
H
H
Iwano
H
H
Yokota
S
S
Ohgiya
A
A
Yuasa
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence / Q000235:genetics; D004795:Enzyme Stability; D005470:Fluorometry; D015870:Gene Expression; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism*; D015151:Immunoblotting; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature; D013997:Time Factors
D001224:Aspartic Acid; C110655:UDP-glucuronosyltransferase, UGT1A6; D014453:Glucuronosyltransferase