HALO ENOL LACTONES - STUDIES ON THE MECHANISM OF INACTIVATION OF ALPHA-CHYMOTRYPSIN
HALO ENOL LACTONES - STUDIES ON THE MECHANISM OF INACTIVATION OF ALPHA-CHYMOTRYPSIN
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DOI：
10.1021/bi00354a037
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发表时间：
1986-03-25
期刊：
影响因子：
2.9
通讯作者：
KATZENELLENBOGEN, JA
中科院分区：
文献类型：
--
作者：
DANIELS, SB;KATZENELLENBOGEN, JA
在以前的调查[丹尼尔斯，S。B.，Cooney，E.，索菲亚，M. J.，Chakravarty，P. K.，和Katzenellenbogen，J. A.（1983）J.Biol.Chem.258，15046-15053]，我们证明了α-芳基取代的五元环和六元环卤代烯醇内酯是胰凝乳蛋白酶的有效抑制剂，并且我们提出它们通过酶激活机制反应：酰基转移到活性位点丝氨酸产生卤代甲基酮，该卤代甲基酮保持束缚在催化位点中，直到其烷基化可接近的亲核残基。在这项研究中，我们更详细地研究了α-胰凝乳蛋白酶灭活的过程。萘基取代的五元和六元溴烯醇内酯。这两种化合物的灭活似乎是活性位点定向的，因为竞争底物延迟了时间依赖性灭活。通过比较内酯与溴甲基酮酸水解产物的灭活效率，研究了可能涉及的灭活副催化机制（产生游离的，而不是活性位点结合的灭活物质）。衍生自五元内酯的溴甲基酮是无效的，而衍生自六元内酯的溴甲基酮是高效的。然而，可能参与的游离酮酸在胰凝乳蛋白酶失活的六元内酯被排除的实验，涉及选择性清除。胰凝乳蛋白酶的长期失活需要溴取代基的存在，并且似乎涉及烷基化而不是酰化反应（抗肼）。此外，1：1的内酯：酶的化学计量证明与14 C-标记的六元内酯。这些结果与先前提出的基于机制的灭活工艺一致。
In a previous investigation [Daniels, S. B., Cooney, E., Sofia, M. J., Chakravarty, P. K., and Katzenellenbogen, J. A. (1983) J. Biol. Chem. 258, 15046-15053], we demonstrated that .alpha.-aryl-substituted five- and six-membered ring halo enol lactones were effective inhibitors of chymotrypsin, and we proposed that they reacted by an enzyme-activated mechanism: acyl transfer to the active site serine generates a halomethyl ketone that remains tethered in the catalytic site until it alkylates an accessible nucleophilic residue. In this study, we have investigated in greater detail the process of chymotrypsin inactivation by an .alpha.-naphthyl-substituted five- and six-membered bromo enol lactone. Inactivation by both compounds appears to be active site directed, since the time-dependent inactivation is retarded by competing substrate. The possible involvement of a paracatalytic mechanism for inactivation (generation of a free, rather than active site bound, inactivating species) was investigated by comparing the inactivation efficiencies of the lactones with that of the bromomethyl keto acid hydrolysis products. The bromomethyl ketone derived from the five-membered lactone is ineffective, whereas that derived from the six-membered lactone is highly efficient. However, the possible involvement of the free keto acid in chymotrypin inactivation by the six-membered lactone is ruled out by experiments involving selective scavenging. The long-term inactivation of chymotrypsin requires the presence of the bromine substituent and appears to involve an alkylation rather than an acylation reaction (hydrazine resistant). Furthermore, a 1:1 lactone:enzyme stoichiometry is demonstrated with the 14C-labeled six-membered lactone. These results are consistent with the mechanism-based inactivation process previously presented.