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|Title:||Effects of amino acid substitutions at positions 33 and 37 on UDP-glucuronosyltransferase 1A9 (UGT1A9) activity and substrate selectivity|
Benjamin C. Lewis
Peter I. MacKenzie
John O. Miners
Flinders Medical Centre
|Keywords:||Biochemistry, Genetics and Molecular Biology;Pharmacology, Toxicology and Pharmaceutics|
|Citation:||Biochemical Pharmacology. Vol.84, No.11 (2012), 1511-1521|
|Abstract:||UGT1A9 contributes to the glucuronidation of numerous drugs and xenobiotics. There is evidence to suggest that the Met33Thr substitution, as occurs in the polymorphic variant UGT1A9*3, variably affects xenobiotic glucuronidation. The equivalent position in UGT1A4 is also known to influence enzyme activity, whilst an N-terminal domain histidine (His37 in UGT1A9) is believed to function as the catalytic base in most UGT enzymes. To elucidate the roles of key amino acids and characterise structure-function relationships, we determined the effects of amino acid substitutions at positions 33 and 37 of UGT1A9 on the kinetics of 4-methylumbelliferone (4-MU), mycophenolic acid (MPA), propofol (PRO), sulfinpyrazone (SFZ), frusemide (FSM), (S)-naproxen (NAP) and retigabine (RTB) glucuronidation, compounds that undergo glucuronidation at either a phenolic (4-MU, MPA, PRO), carboxylate (FSM, NAP), acidic carbon (SFZ) or amine (RTB) function. Substitution of Met33 with Val, Ile, Thr, and Gln, as occur in UGT1A1, UGT1A3, UGT1A4 and UGT1A6 respectively, variably affected kinetics and catalytic efficiency. Whilst K m values were generally higher and V max and CL int values were generally lower than for wild-type UGT1A9 with most substrate-mutant pairs, the pattern and the magnitude of the changes in each parameter differed substantially. Moreover, exceptions occurred; CL int values for MPA and FSM glucuronidation by the position-33 mutants were the same as or higher than that of UGT1A9. Mutation of His37 abolished activity towards all substrates, except RTB N-glucuronidation. The data confirm the importance of single amino acids for UGT enzyme activity and substrate selectivity, and support a pivotal role for residue-33 in facilitating substrate binding to UGT1A9. © 2012 Elsevier Inc.|
|Appears in Collections:||Scopus 2011-2015|
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