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，具有LTA4水解酶活性和氨基肽酶活性。这两种催化活性表现出不同的动力学特性，这些特性与pH依赖性和选择性刺激作用对氨基肽酶活性的选择性作用。在抑制剂实验中，白细胞硫醇对两种催化活性均表现出竞争性抑制作用，而Bestatin对LTA4水解酶活性和对氨基肽酶活性的竞争性抑制作用，这表明LTA4的结合位点对LTA4的结合位点和肽对酶的影响不足。酶与N-乙酰咪唑的乙酰化。这两种催化活性都被这种修饰灭活，通过用净羟胺的心形恢复了冷的催化活性。 Frthermore，这两种活动都可以通过Bestatin保护，免受失活。这些结果表明，位于BESTATIN结合位点或附近的Tyr-OR Cys-残留物的乙酰化造成了两种催化活性的失活。由酶乙酰化引起的紫乙酰基-TYR的紫外分光体定量表明，通过Bestatin保护了1.7-TYR - 替代物，免受乙酰化的保护。用DTNB滴定硫酸基团在天然和乙酰化酶中表明9-SH-沉积物的存在，表明N-乙酰咪唑在酶中均未乙酰化的Cys-乙酰化cys分析。考虑到这些结果，位于Bestatin结合位点或附近的2-TYR分散物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与这些Tyr-ersises的功能特性并不相同，在每次催化中可能会有所不同。