CHEMICAL MODIFICATION OF HUMAN UDP-GLUCURONOSYLTRANSFERASE UGT1(ASTERISK)6 BY DIETHYL PYROCARBONATE - POSSIBLE INVOLVEMENT OF A HISTIDINE RESIDUE IN THE CATALYTIC PROCESS

Chemical modification with diethyl pyrocarbonate (DEPC) of the recombi nant human Liver UDP-glucuronosyltransferase UGT16 in enriched membra ne fractions from a V79 cell line resulted in a rapid inactivation of the glucuronidation reaction, measured with 4-methyl-umbelliferone as aglycone substrate, with a second-order rate constant of 3110 M(-1) mi n(-1) at pH 6.0 and 25 degrees C. The enzymatic activity was restored by hydroxylamine. Chemical modification with 0.2 mM DEPC for 60 s decr eased the apparent V-max 2.4-fold without significantly affecting the apparent K-m toward 4-methylumbelliferone and UDP-glucuronic acid. Sim ilarly, the binding of the photoactivatable cosubstrate analog [beta-P -32]5-azido-UDP-glucuronic acid to the active site was not affected by the chemical modification. The enzyme was protected against this inac tivation by 4-methylumbelliferone, suggesting that the modified residu e was located in or near the aglycone binding site. In contrast, the c osubstrate UDP-glucuronic acid potentiated the irreversible inhibition , indicating a conformational change in the protein upon binding. The pH-dependence of the inactivation was in agreement with the modificati on of an amino acid residue with a pK(a) of 6.1. On the other hand, an alysis of the variation of V-max and V-max/K-m values of the glucuroni dation reaction as a function of the pH revealed the presence of two e ssential residues with a pK(a) within the range 5.7-6.0. The data of t he chemical modification of the recombinant enzyme together with that of the pH dependence of the activity strongly suggest the involvement of a histidine residue, highly reactive toward DEPC, which could be th e base catalyst of the glucuronidation reaction supported by human UGT 16. (C) 1994 Academic Press, Inc. -