Modification of leukotriene A(4) hydrolase/aminopeptidase by sulfhydryl-blocking reagents: Differential effects on dual enzyme activities by methyl-methane thiosulfonate

The presence of a cysteine residue at or near the active site of leukotrien e A(4) hydrolase (EC 3.3.2.6) was suggested by inactivation of the enzyme w ith sulfhydryl-blocking reagents and by protection against inactivation aff orded by substrates and competitive inhibitors. The aminopeptidase activity was more susceptible to inactivation than the epoxide hydrolase activity. The sulfhydryl-modifying reagent methylmethane thiosulfonate reacted with o ne thiol as judged by kinetic data and titration with 5,5'-dithiobis-2-nitr obenzoate, Inactivation was a time- and dose-dependent process of apparent pseudo-first-order and maximal at 80-85%, The inactivation rate was nonsatu rable and strongly influenced by ion strength. The second-order rate consta nt increased from 0.9 to 4.3 M-1 s(-1) in the presence of 0.2 M NaCl. Album in, a stimulator of the aminopeptidase activity, increased apparent inactiv ation rates by shifting pK(a) for the modification from 8.2 to 7.8. The ina ctivated enzyme partially regained activity upon treatment with P-mercaptoe thanol, Peptide substrates and competitive inhibitors protected against ina ctivation. Bestatin, a competitive inhibitor, afforded complete protection with a K-D = 0.15 mu M, similar to K-i = 0.17 mu M for inhibition of peptid ase activity, Treated enzyme had an unchanged K-m but a reduced V-max. The epoxide hydrolase activity was only weakly affected by methylmethane thiosu lfonate with a maximal inactivation of 15-20% after prolonged treatment, Pr etreatment of leukotriene A(4) hydrolase with the reagent did not protect a gainst mechanism-based inactivation by its lipid substrate, leukotriene A(4 ). On the other hand, leukotriene B-4 was a competitive inhibitor of aminop eptidase activity and protected against modification by methyl-methane thio sulfonate. Our results suggest the presence of a cysteine at or close to su bsite S-1' of the active site of leukotriene A(4) hydrolase and that modifi cation of this residue interferes with the function of the aminopeptidase a ctivity, but not the epoxide hydrolase activity. This is the first report t o distinguish the two catalytic activities of leukotriene A(4) hydrolase by chemical means. (C) 1999 Academic Press.