Inhibition of carboxypeptidase A by aldehyde and ketone substrate analogues.
Inhibition of carboxypeptidase A by aldehyde and ketone substrate analogues.
复制标题
醛和酮底物类似物对羧肽酶 A 的抑制作用。
DOI：
10.1021/bi00304a032
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发表时间：
1984
期刊：
影响因子：
2.9
通讯作者：
Kortylewicz,ZP
中科院分区：
文献类型：
--
作者：
Galardy,RE;Kortylewicz,ZP
Richard E. Galardy * 和Zbigniew P. Kortylewicz摘要：DL-2-Benzyl-3-formylpropanoic acid（XIVb）是羧肽酶A的竞争性抑制剂，在50 mM Tris缓冲液（0.5 M氯化钠）中，以0-（反式-对-氯肉桂酰基）-L-β-苯基乳酸盐为底物，pH 7.5时，表观K值为0.48 µ。在pH 7.5的氧化氘中，DL-2-苄基-3-甲酰基丙酸以75%游离醛和25%水合醛的平衡混合物存在。与酶结合的物质可以是游离醛或水合物。因此，结合物质的K_i明显小于观测到的K_i（0.48 µ）。该醛的醇和二氧戊环类似物，dl-2-苄基-4-羟基丁酸（XI）和2-苄基-4，4-二氧戊环类似物，醛和卤甲基酮通常是已知使用酰基酶中间体如弹性蛋白酶的蛋白酶的强可逆底物（Thompson，1973）和木瓜蛋白酶（刘易斯& Wolfenden，1977 a）以及某些氨基肽酶（Birch等人，1972; Kettner等人，1974;安德森等人，1982年）。抑制机制可能涉及在羰基碳原子上添加亲核试剂，将其转化为四面体构型，类似于与酰胺底物水解过渡态相关的中间体（刘易斯& Wolfenden，1977 b）。胰蛋白酶和不可逆抑制剂苄氧羰基[2 - 13C]赖氨酸氯甲基酮之间的四面体加成复合物（Malt-house等人，1983）和胃蛋白酶与碳-13标记的酮之间（Rich等人，1982）已经通过碳-13 NMR观察到。1在胃蛋白酶的情况下，四面体复合物中涉及的亲核试剂的身份尚未确定。它可能是一个水分子或酶结合的亲核试剂。锌金属蛋白酶羧肽酶A被卤甲基酮7V-（溴乙酰基）-7V-甲基苯丙氨酸（Hass & Neurath，1971 a）不可逆地抑制，伴随着谷氨酸-270的烷基化（Hass & Neurath，1971 b）。底物苯甲酰基-L-苯丙氨酸的酮亚甲基类似物，（-）-3-（对甲氧基苯甲酰基）-2-苄基丙酸[（-）-!]，据报道，可可逆地抑制羧肽酶A，Ki为110 µ（Sugimoto和Kaiser，1978）。所报告的关于该酮的研究并未表明抑制机制是否涉及亲核试剂与酮羰基碳可逆加成以产生四面体加合物。然而，在羧肽酶的活性部位发现一个碱催化酮的一个α质子的立体专一性交换，并且该碱被认为是谷氨酸-270或介入的水分子（Sugimoto & Kaiser，1978）。血管紧张素转化酶的三肽底物的酮亚甲基类似物来自肯塔基州大学生物化学系和Sanders-Brown衰老研究中心，Lexington，肯塔基州40536。1983年6月8日接收，1983年11月9日收到修订稿。该项目得到了美国国立卫生研究院赠款HL 27368和RR 05374的支持。
Richard E. Galardy* and Zbigniew P. Kortylewicz abstract: DL-2-Benzyl-3-formylpropanoic acid (XlVb) is a competitive inhibitor of carboxypeptidase A with an apparent K¡ of 0.48 µ at pH 7.5 in 50 mM Tris buffer-0.5 M in sodium chloridewith 0-(lrans-p-chlorocinnamoyl)-L-/3-phenyllactate as substrate. At pH 7.5 in deuterium oxide, DL-2-benzyl-3-formylpropanoic acid exists as an equilibrium mixture of 75% free aldehyde and 25% hydrated aldehyde. The species that binds to the enzyme may be either the free aldehyde or the hydrate. Therefore, the K, of the species bound is significantly less than the observed K¡ of 0.48 µ. The alcohol and dioxolane analogues of this aldehyde, dl-2-benzyl-4-hydroxybutanoic acid (XI) and 2-benzyl-4, 4-(ethylenedioxy) butanoic acid (XXVII), are only weak inhib-jAldehydes and halomethyl ketones are frequently strong reversible inhibitorsof proteases known to employ an acyl enzyme intermediate such as elastase (Thompson, 1973) and papain (Lewis & Wolfenden, 1977a) and of certain amino-peptidases (Birch et al., 1972; Kettner et al., 1974; Anderson et al., 1982). The mechanism of inhibition probably involves the addition of a nucleophile to the carbonyl carbon atom, converting it to a tetrahedral configuration which resembles intermediates related to the transition state for amide substrate hydrolysis (Lewis & Wolfenden, 1977b). Tetrahedral addition complexes between trypsin and the irreversible inhibitor benzyloxycarbonyl [2-13C] lysine chloromethyl ketone (Malt-house et al., 1983) and between pepsin and a carbon-13 labeled ketone (Rich et al., 1982) have been observed by carbon-13 NMR. 1 In the case of pepsin, the identity of the nucleophile involved in the tetrahedral complex was not established. It could be a water molecule or an enzyme-bound nucleophile. The zinc metalloprotease carboxypeptidase A is irreversibly inhibited by the halomethyl ketone 7V-(bromoacetyl)-7V-methylphenylalanine (Hass & Neurath, 1971a) accompanied by alkylation of glutamic acid-270 (Hass & Neurath, 1971b). The ketomethylene analogue of the substrate benzoyl-L-phenylalanine,(-)-3-(p-methoxybenzoyl)-2-benzylpropanoic acid [(-)-!], was reported to reversibly inhibit carboxypeptidase A with a K, of 110 µ (Sugimoto and Kaiser, 1978). The reported studies with this ketone do not suggest whether the mechanism of inhibition involves reversible addition of a nu-cleophile to the ketone carbonyl carbon to produce a tetra-hedral adduct. However, a base atthe active site of carbox-ypeptidase was found to catalyze stereospecific exchange of one of the a protons of the ketone, and this base was proposed to be glutamic acid-270 or an intervening water molecule (Sugimoto & Kaiser, 1978). A ketomethylene analogue of a tripeptide substrate of angiotensin-converting enzyme has f From the Department of Biochemistry and the Sanders-Brown Research Center on Aging, University of Kentucky, Lexington, Kentucky 40536. Received June 8, 1983·, revised manuscript received November 9, 1983. This project was supported by National Institutes of Health Grants HL 27368 and RR 05374.