Kinetic and stereochemical studies on novel inactivators of C-terminal amidation
Kinetic and stereochemical studies on novel inactivators of C-terminal amidation
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DOI：
10.1042/0264-6021:3500521
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发表时间：
2000-09-01
影响因子：
4.1
通讯作者：
May, SW
中科院分区：
文献类型：
--
作者：
Feng, J;Shi, J;May, SW
C-末端酰胺化是许多神经肽生物活化所需的翻译后修饰，需要肽基甘氨酸单加氧酶（PAM，EC 1.14. 17.3）和肽酰氨基乙醇酸裂解酶（PGL，EC 4.3.2.5）。在这里，我们介绍了新的化合物，其中烯烃功能被纳入肽类似物作为最有效的营业额依赖性灭活剂的PAM。采用传统的稀释法和更复杂的过程曲线法，获得了4-氧代-5-乙酰氨基-6-苯基-2-己烯酸和4-氧代-5-乙酰氨基-6-（2-噻吩基）-2-己烯酸灭活PAM的动力学参数。从进展曲线法获得的结果表明，这些化合物表现出纯竞争性灭活剂的动力学特征（即在灭活过程中不形成ESI复合物）。基于k（inaet）/Ki值，4-氧代-5-乙酰氨基-6-（2-噻吩基）-己-2-烯酸比苯甲酰基丙烯酸酯（一种缺乏肽部分的化学上类似的烯烃灭活剂）几乎强两个数量级。立体化学研究证实，通过4-氧代-5-乙酰氨基-6-（2-噻吩基）-己-2-烯酸的PAM灭活相对于P-2位的部分是立体特异性的，这与先前底物和可逆抑制剂的结果一致。相比之下，2，4-二氧代-5-乙酰氨基-6-苯基己酸（其是抗坏血酸的竞争性抑制剂）在结合PAM和多巴胺-β-羟化酶的抗坏血酸位点时表现出低程度的立体特异性。
C-terminal amidation, a required post-translational modification for the bioactivation of many neuropeptides, entails sequential enzymic action by peptidylglycine il-mono-oxygenase (PAM, EC 1.14. 17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). Here we introduce novel compounds in which an olefinic functionality is incorporated into peptide analogues as the most potent turnover-dependent inactivators of PAM. Kinetic parameters for PAM inactivation by 4-oxo-5-acetamido-6-phenyl-hex-2-enoic acid and 4-oxo-5-acetamido-6-(2-thienyl)-hex-2-enoic acid were obtained by using both the conventional dilution assay method and the more complex progress curve method. The results obtained from the progress curve method establish that these compounds exhibit the kinetic characteristics of pure competitive inactivators (i.e. no ESI complex forms during inactivation). On the basis of k(inaet)/K-i values, 4-oxo-5-acetamido-6-(2-thienyl)-hex-2-enoic acid is almost two orders of magnitude more potent than benzoylacrylate, a chemically analogous olefinic inactivator that lacks the peptide moiety. Stereochemical studies established that PAM inactivation by 4-oxo-5-acetamido-6-(2-thienyl)-hex-2-enoic acid is stereo-specific with respect to the moiety at the P-2 position, which is consistent with previous results with substrates and reversible inhibitors. In contrast, 2,4-dioxo-5-acetamido-6-phenylhexanoic acid, which is a competitive inhibitor with respect to ascorbate, exhibits a low degree of stereospecificity in binding to the ascorbate sites of both PAM and dopamine-beta-hydroxylase.