Revised Manuscript Received December 9, 1994s abstract: We have employed chemical modification to identify amino acids essential for the catalytic activities of the bifunctional zinc metalloenzyme leukotriene A4 hydrolase (EC 3.3. 2.6). The epoxide hydrolase and the peptidase activity were both rapidly inactivated by jV-acetylimidazole and tetrani-tromethane. Furthermore, treatment with 2, 3-butanedione and phenylglyoxal also resulted in loss of both activities. Leukotriene A4 hydrolase could be protected from inactivation by these tyrosyl and arginyl reagents by the competitive inhibitors bestatinand captopril, respectively. Two tyrosyl and three arginyl residues were found by differential labeling techniquesto be protected by the inhibitors, which thus suggested that these amino acids are located close to or at the active center of the enzyme. Limited modification by thiol reagents and particularly methyl methanethiosulfonate led to a> 10-fold increase in the peptidase activity and a decreased epoxide hydrolase activity, whereas prolonged treatment inhibited both activities. Kinetic analysis of modified enzyme, using the substrate alanine p-nitroanilide, revealed that the stimulatory effect on the peptidase activity was due to increased enzyme turnover, whereas the Michaelis constant remained unaffected. Furthermore, the modified enzyme displayed a reduced apparent affinity constant for chloride ions, which strongly stimulate the peptidase activity. Neither activation nor inactivation by methyl methanethiosulfonate was influenced by the presence of competitive inhibitors, which suggested that this compounddid not react with amino acids at the active center but rather with residues of importance for the overall enzyme conformation.

Leukotriene (LT) 1A4 hydrolase (EC 3.3. 2.6) is a bifunc-tional zinc metalloenzyme (Haeggstrom et al., 1990; Minami et al., 1990) which catalyzes the hydrolysis of the unstable epoxide intermediate LTA» into the proinflammatory substance leukotriene B4 (LTB4) andalso exhibits an anúdase/peptidase activity toward synthetic substrates. Leukotriene B4 is a potent chemotactic agent for leukocytes and stimulates a number of other leukocyte functions, including aggregation, enhancement of lysosomal enzyme release, superoxide anion production, enhancement of complement-dependent cyto-toxicity reactions, and leukocyte adherence to the endothe-lium (Ford-Hutchinson, 1990). Since there is evidence for increased LTB4 formation in human inflammatory diseases, this compound has the potential for being an important mediator in a number of disease states (Fretland et al., 1990). The physiological significance of the aminopeptidase activity is largely unclear and remains to be evaluated. The naturally occurring enkephalins are substrates in vitrofor this activity; however, their hydrolysis is inefficient compared with that of LTA4 (Griffin et al., 1992). Recent data suggest that LTA4 hydrolase is an arginine aminopeptidase with high efficiency