Selective inactivation of parvulin-like peptidyl-prolyl cis/trans isomerases by juglone
Selective inactivation of parvulin-like peptidyl-prolyl cis/trans isomerases by juglone
复制标题
DOI：
10.1021/bi973162p
复制
发表时间：
1998-04-28
期刊：
影响因子：
2.9
通讯作者：
Fischer, G
中科院分区：
文献类型：
--
作者：
Hennig, L;Christner, C;Fischer, G
与FK506结合蛋白和亲环素相反，肽基-脯氨酰顺式/反式异构酶（PPI酶; E. C. 5.2.1.8 we.已经发现胡桃醌，5-羟基-1，4-萘醌，不可逆地抑制几种小蛋白的酶活性，如E. coli parvulin、酵母菌PITU1/Ptf1和人Pin1，从而允许在其它PPIases存在下选择性灭活这些酶。通过对E. coli parvulin及其Cys69Ala变体与胡桃醌的结合。对于所有研究的小蛋白，通过缓慢过程获得完全灭活，其特征在于伪一级速率常数在5.3 x 10（-4）至4.5 x 10（-3）s（-1）范围内。失活的小蛋白含有两个胡桃醌分子，由于巯基与胡桃醌的Michael加成，所述胡桃醌分子共价结合到Cys41和Cys69的侧链。氧化还原反应没有贡献的失活过程。由于巯基修饰的进行速度比酶失活速度快5倍，因此它被认为是失活的必要条件，但不是充分条件。当通过远紫外圆二色性（CD）测量时，巯基修饰后的结构改变速率与失活速率完全平行。因此，认为小蛋白活性位点的部分解折叠是PPIase活性恶化的原因。
In contrast to FK506 binding proteins and cyclophilins, the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases; E.C. 5.2.1.8) cannot be inhibited by either FK506 or cyclosporin A. We. have found that juglone, 5-hydroxy-1,4-naphthoquinone, irreversibly inhibits the enzymatic activity of several parvulins, like the E. coli parvulin, the yeast Ess1/Ptf1, and human Pin1, in a specific manner, thus allowing selective inactivation of these enzymes in the presence of other PPIases. The mode of action was studied by analyzing the inactivation kinetics and the nature of products of the reaction of E. coli parvulin and its Cys69Ala variant with juglone. For all parvulins investigated, complete inactivation was obtained by a slow process that is characterized by pseudo-first-order rate constants in the range of 5.3 x 10(-4) to 4.5 x 10(-3) s(-1). The inactivated parvulin contains two juglone molecules that are covalently bound to the side chains of Cys41 and Cys69 because of a Michael addition of the thiol groups to juglone. Redox reactions did not contribute to the inactivation process. Because thiol group modification was shown to proceed 5-fold faster than the rate of enzyme inactivation, it was considered as a necessary but not sufficient condition for inactivation. When measured by far-UV circular dichroism (CD), the rate of structural alterations following thiol group modification parallels exactly the rate of inactivation. Thus, partial unfolding of the active site of the parvulins was thought to be the cause of the deterioration of PPIase activity.