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

LTA 4水解酶是一种具有LTA 4水解酶活性和氨肽酶活性的双功能酶。这两种催化活性表现出不同的动力学性质，包括pH依赖性和Cl对氨肽酶活性的选择性刺激作用。在抑制剂实验中，亮氨硫醇对两种酶的催化活性均表现出竞争性抑制，而bestatin对LTA 4水解酶活性表现出非竞争性抑制，对氨肽酶活性表现出竞争性抑制，这表明LTA 4与酶上多肽的结合位点并不完全相同。这两种催化活性都被这种修饰失活，可以通过用中性羟胺处理来恢复。然而，这两种活性都可以被bestatin保护免于失活。这些结果表明，位于bestatin结合位点或附近的Tyr-或Cys-残基的乙酰化是导致这两种催化活性失活的原因。由酶的乙酰化产生的O-乙酰基-Tyr的UV荧光定量表明，1.7-Tyr-残基被bestatin保护免于乙酰化。用DTNB滴定巯基表明，在天然酶和乙酰化酶中均存在9-SH-残基，表明N-乙酰咪唑没有乙酰化酶中的Cys-残基。考虑到这些结果，位于bestatin结合位点中或附近的2-Tyr-残基的乙酰化导致两种催化活性的损失。由于两种催化活性的底物结合位点不相同，因此这些Tyr残基的功能性质在每种催化中可能不同。

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.