RT Journal Article SR Electronic T1 Importance of Histidine Residues for the Function of the Human Liver UDP-Glucuronosyltransferase UGT1A6: Evidence for the Catalytic Role of Histidine 370 JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 1609 OP 1615 DO 10.1124/mol.58.6.1609 VO 58 IS 6 A1 Mohamed Ouzzine A1 Laurence Antonio A1 Brian Burchell A1 Patrick Netter A1 Sylvie Fournel-Gigleux A1 Jacques Magdalou YR 2000 UL http://molpharm.aspetjournals.org/content/58/6/1609.abstract AB The human UDP-glucuronosyltransferase isoform UGT1A6 catalyzes the nucleophilic attack of phenolic xenobiotics on glucuronic acid, leading to the formation of water-soluble glucuronides. Based on the irreversible inhibition of the enzyme activity by the histidyl-selective reagent diethyl pyrocarbonate (DEPC), histidine was suggested to play a key role in the glucuronidation reaction. Therefore, the role of four strictly conserved histidine residues (His38, His361, His370, and His485) in the glucuronidation of 4-methylumbelliferone, as reporter substrate, was examined using site-directed mutagenesis. For this purpose, stable heterologous expression of wild-type and mutant UGT1A6 was achieved in the yeastPichia pastoris. Replacement of histidine residues by alanine or glutamine led to fully inactive H38A, H38Q, and H485A mutants. Substitution of His361 by alanine affected the interaction of the enzyme with the cosubstrate, as indicated by a 4-fold increase in the K m value toward UDP-glucuronic acid. Interestingly, H370A mutant presented a severely impaired catalytic efficiency (with a V max value approximately 5% that of the wild-type), whereas conservative substitution of His370 by glutamine (H370Q) led to a significant restoration of activity. Whereas H361A was inactivated by DEPC as the wild-type enzyme, this chemical reagent only produced a minor effect on either H370Q or H370A mutant, providing evidence that His370 is probably the reactive histidine residue targeted by DEPC. The dramatic changes in catalytic efficiency on substitution of His370 by alanine and the ability of glutamine to function in place of histidine along with a weak sensitivity of these mutants to DEPC strongly suggest that His370 plays a catalytic role in the glucuronidation reaction.