Inhibition of trypsin with active-site-directed enzyme-activated nitrosoamide substrates.
Inhibition of trypsin with active-site-directed enzyme-activated nitrosoamide substrates.
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用活性位点定向酶激活的亚硝酰胺底物抑制胰蛋白酶。
DOI：
10.1021/bi00046a019
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Chen,Y
中科院分区：
文献类型：
--
作者：
White,EH;Chen,Y
一系列活性位点定向酶激活的胰蛋白酶亚硝酰胺抑制剂已被设计、合成和测试。该抑制剂含有可产生烷化剂的N-亚硝基酰胺基团和在脂肪族碳链末端的带正电荷的铵离子基团，以提供特异性。系列1中的化合物在正常条件下的抑制半衰期为0.6至2分钟。其中一种化合物，N-(4-氨基-1-丁基)-N-亚硝基苯甲酰胺(Ib)，是一种非常有效的抑制剂；其分配比 fo/kinact 为零，表明它可能是胰蛋白酶和相关酶的有用滴定剂。竞争性抑制剂苯甲脒大大降低了抑制程度，表明抑制剂在活性位点起作用。注意到两种抑制模式：可逆和不可逆。 N-亚硝酰胺抑制剂在主要特异性的引导下与胰蛋白酶结合袋结合。然后，他们酰化酶（Ser-195），产生离去基团，在活性位点产生重氮离子（或）碳正离子；它们与酶的近端羧酸侧链反应形成羧酸酯，可能是 Asp-194 的酯。如果烷基上存在伯氨基，则酯（R¡ COO13CH2-(CH2)„NH2 的 13C NMR < 5 67.2 ppm）通过 O—-N 酰基迁移重排成酰胺形式（RiCONH (CH2)„13CH20H 的 13C NMR - 62.9 ppm）；产生不可逆的酶抑制。基于位点特异性基团方向的模型提出了胰蛋白酶活性位点上的/V-(4-氨基-1-丁基)-/V-亚硝基-/V'-异丁酰-D-丙氨酰胺(D2a)及其对映体来解释D-异构体对胰蛋白酶的优先抑制作用，分析揭示了决定抑制剂的抑制模式的四个因素：1、烷基的长度；2、酰基酶的稳定性；4、选项0~N酰基
A series of active-site-directed enzyme-activated nitrosoamide inhibitors of trypsin has been designed, synthesized, and tested. The inhibitors contain an/V-nitrosoamide group that can generate an alkylating agent and a positively charged ammoniumion group at the end of an aliphatic carbon chain that provides specificity. The half-lives of inhibition under normal conditions were 0.6 to2 min for compounds in series 1. One of the compounds,/V-(4-amino-l-butyl)-/V-nitrosobenzamide (lb), is a very efficient inhibitor; its partition ratio, fo/kinact, is zero suggesting that it may be a useful titrant for trypsin and related enzymes. The extent of inhibition is substantially decreased by the competitive inhibitor benzamidine, indicating that the inhibitors were operating in the active site. Two modes of inhibition were noted: reversible and irreversible. The/V-nitrosoamide inhibitors bind to the trypsin binding pocket guided by the primary specificity. They then acylate the enzyme (at Ser-195), producing a leaving group that generates diazonium ions (or) carbocations in the active site; these react with a proximal carboxylic acid side chain of the enzyme to form a carboxylic acid ester, presumably that of Asp-194. If primary amino groups are present on the alkyl group, the ester (13C NMR< 5 67.2 ppm for R¡ COO13CH2-(CH2)„NH2) rearranges into the amide form (13C NMR ó 62.9 ppm for RiCONH (CH2)„13CH20H) through an O—-N acyl migration; an irreversibly inhibited enzyme results. A model based on the orientation of the site-specific group of/V-(4-amino-l-butyl)-/V-nitroso-/V'-isobutyryl-D-alaninamide (D2a) and its l antipode in the active site of trypsin is proposed to explain the preferential inhibition of trypsin by the D-isomer. Analysis of the structure—inhibition relationship revealed four factors that determinethe inhibition modes of the inhibitors: 1, length of the alkyl group; 2, stability of the acyl-enzyme; 3, the option 0~ N acyl