Chemical modification of leukotriene (LT) A4 hydrolase and structral analysis of its active centers.

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LTA4 hydrolase is a bifunctional enzyme which has both LTA4 hydrolase activity and aminopeptidase activity. These two catalytic activities showed different kinetic properties inclding pH dependencies and selective stimlatory effect of Cl on aminopeptidase activity. In inhibitor experiments, leucinthiol exhibited competitive inhibition on both catalytic activities, while bestatin showed uncompetitive inhibition on LTA4 hydrolase activity and competitive inhibition on aminopeptidase activity, which indicates that the binding sites of LTA4 and peptides on the enzyme is not identical.As a means of identifying amino acid residues contributing to catalytic activities, we performed acetylation of the enzyme with N-acetylimidazole. Both catalytic activities were inactivated by this modification, which cold be recovered by the tretment with netral hydroxylamine. Frthermore, both activities could be protected from inactivation by bestatin. These results sggested that acetylation of Tyr-or Cys-residues located in or near the bestatin binding site was responsible for the inactivation of both catalytic activities. The UV spectrophotometrical quantification of O-acetyl-Tyr resulting from acetylation of the enzyme indicated that 1.7-Tyr-residues were protected from acetylation by bestatin. Titration of sulfydoryl groups with DTNB showed the presence of 9-SH-residues in both native and acetylated enzyme indicating that N-acetylimidazole did not acetylated Cys-residues in the enzyme. Considering these results, acetylation of 2-Tyr-residues located in or near the bestatin binding site resulted in the loss of both catalytic activities. As substrates binding sites for the two catalytic activities are not identical, functional properties of these Tyr-resides may be different in each catalysis.