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The alpha-amidating enzyme. The intermediary reaction steps and the structure/function relationship as a bifunctional enzyme.

α-酰胺化酶。

基本信息

批准号：
05680558
负责人：
NOGUCHI Masato
金额：
$ 1.28万
依托单位：
Kurume University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

Peptidylglycine alpha-amidating activity catalyzes the oxidation of a C-terminally glycine-extended peptide to a desglycine alpha-amidated peptide at the expense of ascorbate and O_2 in the presence of copper. The reaction involves oxidative N-dealkylation within the terminal glycine, retaining the glycine N and releasing the remainder as glyoxylate. We revealed that the reaction consists of two steps via a carbinolamide as an intermediate (peptidyI alpha-hydroxyglycine), and also that two separate enzymes derived from a common precursor protein, peptidylglycine alpha-hydroxylase (PH) and peptidylamidoglycolate lyase (PGL), catalyze these steps, formation of the carbinolamide and its conversion into the alpha-amide and glyoxylate, respectively. The goals of the projects were (1) to clarify whether an alpha-carbon radical of glycine is involved in the formation of the carbinolamide intermediate by investigating isotope effects on the PH reaction, and (2) to construct an expression vecto … More r that is able to secrete the precursor protein into medium in order to probe the structure/function relationship as a bifunctional enzyme. Along with these projects, we have obtained the following results. (1) The hydroxylation of glycine is a ping-pong Bi Bi reaction in terms of ascorbate and the substrate (D-Tyr-Val-Gly). The deuterium effects on V/K and Vmax, using D-Tyr-Val-Gly-2,2-d2, are about 1.2, far less than 5.7 which has been reported by Kizar et al. Much more detailed kinetic study with tritium-labeled substrate is on going in order to learn the true isotope effect values, ^DV/K and ^DVmax. (2) The cDNA library has been prepared from rat atrium. The cDNA clone encoding the precursor protein is in search. (3) We have found the amidating activity in the cerebrospinal fluids (CSF) is increased in patients with multiple sclerosis, especially during the active stage, compared with that in patients with other neurological diseases. This suggests that the amidating activity in CSF could be an indication reflecting the physiological and morbid states of the central nervous system. Less

肽酰甘氨酸α-酰胺化活性催化C-末端甘氨酸延伸肽在铜存在下以抗坏血酸和O_2为代价氧化为脱甘氨酸α-酰胺化肽。该反应涉及末端甘氨酸内的氧化N-脱烷基化，保留甘氨酸N并释放其余的乙醛酸盐。我们发现，该反应由两个步骤组成，通过甲醇酰胺作为中间体（肽基α-羟基甘氨酸），也是两个独立的酶来自一个共同的前体蛋白，肽基甘氨酸α-羟化酶（PH）和肽酰氨基乙醇酸裂解酶（PGL），催化这些步骤，甲醇酰胺的形成和转化为α-酰胺和乙醛酸，分别。这些项目的目标是：（1）通过研究同位素对PH反应的影响，阐明甘氨酸的α-碳自由基是否参与了甲醇酰胺中间体的形成;（2）构建表达载体， ...更多信息 R，其能够将前体蛋白分泌到培养基中，以作为双功能酶来探测结构/功能关系。沿着这些项目，我们取得了以下成果。(1)甘氨酸的羟基化反应是一个以抗坏血酸和底物（D-Tyr-Val-Gly）为基础的乒乓式Bi Bi反应。采用D-Tyr-Val-Gly-2，2-d2，氘对V/K和Vmax的影响约为1.2，远小于Kizar等人报道的5.7。为了了解真实的同位素效应值^DV/K和^DVmax，正在进行更详细的氚标记底物动力学研究。(2)从大鼠心房制备cDNA文库。编码前体蛋白的cDNA克隆正在寻找中。(3)我们发现，与其他神经系统疾病患者相比，多发性硬化患者脑脊液（CSF）中的酰胺化活性增加，尤其是在活动期。这表明脑脊液中的酰胺化活性可能是反映中枢神经系统生理和病理状态的指标。少