Novel inactivators of serine proteases based on 6-chloro-2-pyrone.
Novel inactivators of serine proteases based on 6-chloro-2-pyrone.
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基于 6-氯-2-吡喃酮的新型丝氨酸蛋白酶灭活剂。
DOI：
10.1021/bi00282a034
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发表时间：
1983
期刊：
影响因子：
2.9
通讯作者：
Abeles,RH
中科院分区：
文献类型：
--
作者：
Westkaemper,RB;Abeles,RH
理查德B。Westkaemper* 和Robert H.研究了丝氨酸蛋白酶（酯酶）与6-氯-2-吡喃酮的相互作用。发现3-和5-苄基-6-氯-2-吡喃酮以及3-和5-甲基-6-氯-2-吡喃酮对糜蛋白酶、β-溶解蛋白酶、猪肝弹性蛋白酶和胆碱酯酶具有时间依赖性失活。未观察到未取代的6-氯-2-吡喃酮失活。取代的吡喃酮不会使木瓜蛋白酶或羧肽酶A以及许多其他非蛋白水解酶失活。因此，这些取代的氯代吡喃酮对丝氨酸蛋白酶显示出明显的选择性。其中6-氯取代基被H或OH取代的模拟不成立。因此，卤素的存在对于灭活是必不可少的。胰凝乳蛋白酶催化3-苄基-6-氯-2-吡喃酮的水解。在pH 7.5下，（E）-4-苄基-2-戊烯二酸是主要产物，并且2-苄基-2-戊烯二酸酐是次要产物。发现的水解产物与失活的酶分子数的比率在14至40之间变化。用3-苄基化合物灭活的酶不显示吡喃酮环的光谱特征。这表明3-苄基-6-氯-2-吡喃酮的失活在酶促内酯水解后以基于机制的方式发生。当用5-苄基化合物使酶失活时，观察到由于吡喃酮环引起的吸光度。我们认为，失活发生通过一个活性位点定向机制，涉及一个活性位点亲核试剂的吡喃酮环的1，6-共轭加成。自杀底物或基于机制的不可逆灭活剂的概念已经导致了许多酶类别的抑制剂的设计（Abeles和Maycock，1976; Rando，1975; Seiler等人，1978;沃尔什，1982）。这种类型的灭活剂含有潜在的反应性官能团，该官能团来自Brandéis大学生物化学研究生部，Waltham，马萨诸塞州02254。1982年10月12日收到。第1447号出版物。由美国国立卫生研究院（GM 12633）向RHA提供的研究经费和美国国立卫生研究院向RBW提供的博士后奖学金（GM 07939）支持。1现住址：弗吉尼亚联邦大学弗吉尼亚医学院药物化学系，弗吉尼亚州里士满，邮编23219。在酶促活化后仅在活性位点被掩蔽。迄今为止描述的大多数基于机制的不可逆灭活剂是用于吡哆醛依赖性酶的。丝氨酸蛋白酶在许多生化系统和疾病状态中起关键作用（Barrett，1980）。例如，导致肺弹性蛋白破坏的人白细胞弹性蛋白酶与肺气肿的发展有关（Mittman，1972; Turino等人，1974; Hance & Crystal，1975; Boudier等人，1981年）。基于这些原因，我们认为有必要探索开发其他基于机制的丝氨酸蛋白酶不可逆灭活剂的可能性。虽然这项工作正在进行中，Chakravarty等人。（1982）报道发现了一种新型的基于机制的
Richard B. Westkaemper* and Robert H. Abeles* abstract: The interaction of serine protease (esterases) with 6-chloro-2-pyrones was investigated. Time-dependent inac-tivation of chymotrypsin,-lytic protease, pigliver elastase, and cholinesterase was found with 3-and 5-benzyl-6-chloro-2-pyrone, as well as 3-and 5-methyl-6-chloro-2-pyrone. No inactivation was observed with the unsubstituted 6-chloro-2-pyrone. The substituted pyrones did not inactivate papain or carboxypeptidase A, as well as a number of other nonproteolytic enzymes. Thesubstituted chloropyrones, therefore, show considerableselectivity toward serineproteases. Ana-logues in which the 6-chloro substituent is replaced by H or OH do not inactivate. The presence of the halogen is, therefore, essential for inactivation. Chymotrypsin catalyzes the hydrolysis of 3-benzyl-6-chloro-2-pyrone. At pH 7.5,(£)-4-benzyl-2-pentenedioic acid is the major product, and 2-benzyl-2-pentenedioic anhydride is a minor product. The ratio of hydrolysis product found to the number of enzyme molecules inactivated varies from 14 to 40. The enzyme inactivated with the 3-benzyl compound does not show a spec-trum characteristic of the pyrone ring. This suggests that inactivation by 3-benzyl-6-chloro-2-pyrone occurs in a mechanism-based fashion after enzymatic lactone hydrolysis. When the enzyme is inactivated with the 5-benzyl compound, absorbance due to the pyrone ring is observed. We suggest that inactivation occurs through an active site directed mechanism involving a 1, 6-conjugate addition of an active site nucleophile to the pyrone ring. e concept of suicide substrates or mechanism-based irreversible inactivators has led tothe design of inhibitors for a number of enzyme classes (Abeles & Maycock, 1976; Rando, 1975; Seiler et al., 1978; Walsh, 1982). Inactivators of this type contain a latently reactive functional group that is unt From the Graduate Department of Biochemistry, Brandéis Univer-sity, Waltham, Massachusetts 02254. Received October 12, 1982. This is Publication No. 1447. Supported by a research grant to RHA from the National Institutes of Health (GM 12633) and by a National Institutes of HealthPostdoctoral Fellowship to RBW (GM 07939). 1 Present address: Department of Pharmaceutical Chemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23219. masked only at the active site after enzymatic activation. Most mechanism-based irreversible inactivators described to date are for pyridoxal-dependent enzymes. Serine proteases play a crucial role in many biochemical systems and disease states (Barrett, 1980). Human leucocyte elastase, for example, which causes the destruction of lung elastin, has been implicated in the development of emphysema (Mittman, 1972; Turino et al., 1974; Hance & Crystal, 1975; Boudier et al., 1981). For these reasons, we thought it de-sirable to explore the possibility of developing additional mechanism-based irreversible inactivators of serine proteases. While this work was in progress, Chakravarty et al.(1982) reported the discovery of a new type of mechanism-based