Importance of histidine residues for the function of the human liver UDP-glucuronosyltransferase UGT1A6: Evidence for the catalytic role of histidine370

The human UDP-glucuronosyltransferase isoform UGT1A6 catalyzes the nucleoph ilic attack of phenolic xenobiotics on glucuronic acid, leading to the form ation of water-soluble glucuronides. Based on the irreversible inhibition o f the enzyme activity by the histidyl-selective reagent diethyl pyrocarbona te (DEPC), histidine was suggested to play a key role in the glucuronidatio n reaction. Therefore, the role of four strictly conserved histidine residu es (His38, His361, His370, and His485) in the glucuronidation of 4-methylum belliferone, as reporter substrate, was examined using site-directed mutage nesis. For this purpose, stable heterologous expression of wild-type and mu tant UGT1A6 was achieved in the yeast Pichia pastoris. Replacement of histi dine 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 th e K-m value toward UDP-glucuronic acid. Interestingly, H370A mutant present ed a severely impaired catalytic efficiency (with a V-max value approximate ly 5% that of the wild-type), whereas conservative substitution of His370 b y glutamine (H370Q) led to a significant restoration of activity. Whereas H 361A was inactivated by DEPC as the wild-type enzyme, this chemical reagent only produced a minor effect on either H370Q or H370A mutant, providing ev idence that His370 is probably the reactive histidine residue targeted by D EPC. 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 tha t His370 plays a catalytic role in the glucuronidation reaction.