Modification of Leukotriene A4 Hydrolase/Aminopeptidase by Sulfhydryl-Blocking Reagents: Differential Effects on Dual Enzyme Activities by Methyl-Methane Thiosulfonate

Abstract

The presence of a cysteine residue at or near the active site of leukotriene A4 hydrolase (EC 3.3.2.6) was suggested by inactivation of the enzyme with sulfhydryl-blocking reagents and by protection against inactivation afforded by substrates and competitive inhibitors. The aminopeptidase activity was more susceptible to inactivation than the epoxide hydrolase activity. The sulfhydryl-modifying reagent methyl-methane thiosulfonate reacted with one thiol as judged by kinetic data and titration with 5,5′-dithiobis-2-nitrobenzoate. Inactivation was a time- and dose-dependent process of apparent pseudo-first-order and maximal at 80–85%. The inactivation rate was nonsaturable and strongly influenced by ion strength. The second-order rate constant increased from 0.9 to 4.3 M−1 s−1 in the presence of 0.2 M NaCl. Albumin, a stimulator of the aminopeptidase activity, increased apparent inactivation rates by shifting pKa for the modification from 8.2 to 7.8. The inactivated enzyme partially regained activity upon treatment with β-mercaptoethanol. Peptide substrates and competitive inhibitors protected against inactivation. Bestatin, a competitive inhibitor, afforded complete protection with a KD = 0.15 μM, similar to Ki = 0.17 μM for inhibition of peptidase activity. Treated enzyme had an unchanged Km but a reduced Vmax. The epoxide hydrolase activity was only weakly affected by methyl-methane thiosulfonate with a maximal inactivation of 15–20% after prolonged treatment. Pretreatment of leukotriene A4 hydrolase with the reagent did not protect against mechanism-based inactivation by its lipid substrate, leukotriene A4. On the other hand, leukotriene B4 was a competitive inhibitor of aminopeptidase activity and protected against modification by methyl-methane thiosulfonate. Our results suggest the presence of a cysteine at or close to subsite S1′ of the active site of leukotriene A4 hydrolase and that modification of this residue interferes with the function of the aminopeptidase activity, but not the epoxide hydrolase activity. This is the first report to distinguish the two catalytic activities of leukotriene A4 hydrolase by chemical means.