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

白三烯 (LT) A4 水解酶的化学修饰及其活性中心的结构分析。

基本信息

批准号：
07670135
负责人：
OHISHI Nobuya
金额：
$ 1.47万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670135/
关键词：
Leukotriene A4 hydrolase Aminopeptidase Bestatin N-acetylimidazole Active center 化学修飾

项目摘要

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.

LTA4水解酶是一种双功能酶，同时具有LTA4水解酶活性和氨肽酶活性。这两种催化活性表现出不同的动力学特性，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，亮氨酸硫醇对两种催化活性均表现出竞争性抑制，而贝他汀对LTA4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LTA4与肽在酶上的结合位点并不相同。为了鉴定有助于催化活性的氨基酸残基，我们用 N-乙酰咪唑对酶进行乙酰化。这种修饰使两种催化活性失活，通过用中性羟胺处理可以恢复。此外，贝他汀可以保护这两种活性免于失活。这些结果表明位于贝他汀结合位点内或附近的Tyr-或Cys-残基的乙酰化是导致两种催化活性失活的原因。由酶乙酰化产生的 O-乙酰基-Tyr 的紫外分光光度定量表明 1.7-Tyr 残基受到贝他汀的保护而免于乙酰化。用 DTNB 滴定硫酰基显示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，位于贝他汀结合位点内或附近的 2-Tyr 残基的乙酰化导致两种催化活性的丧失。由于两种催化活性的底物结合位点不相同，因此这些酪氨酸残基在每种催化中的功能特性可能不同。