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Study on the Enzymatic Reaction Mechanism catalyzed by Histidine Ammonia-Lyase Using Stable Isotope Methodology

稳定同位素方法研究组氨酸解氨酶催化的酶反应机理

基本信息

批准号：
03807143
负责人：
FURUTA Takashi
金额：
$ 1.15万
依托单位：
Tokyo College of Pharmacy
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

L-Histidine labeled with deuterium at C-5' of the imidazole ring, L-[5'-^2H]histidine, was used as a probe for investigating an enzymatic reaction mechanism in the elimination of ammonia catalyzed by histidine ammonia-lyase (EC 4.3.1.3). The labeled L-histidine was incubated with histidine ammonia-lyase from Pseudomonas fluorescens at pH 7.0 or pH9.0 at 25.0 ﾟC for 24 h. The time course of the reaction was examined to determine the rates of enzyme-catalyzed hydrogen exchange at C-5' of L-histidine and urocanic acid. The finding of the enzyme-catalyzed hydrogen exchange at C-5' of both L-histidine and urocanic acid provided a rational explanation for a stepwise reversible mechanism via a carbanion intermediate in the elimination reaction. The stability of carbanion was also demonstrated to be approximately three times higher at pH 7.0 than at pH 9.0.The deuterium isotope effects for the histidine ammonia-lyase reaction have also been studied under the various pH conditions (pH 7.0-10.5) by using both L-[3,3-^2H_2]histidine and L-[5'-^2H]histidine. Comparison of the Michaelis constants with labeled (L-[3,3-^2H_2]histidine) and unlabeled substrates revealed that the isotope effects on V_<max> and V/K were 1.00-1.38 and 1.18-1.63, respectively, indicating pH dependent. As the pH was increased,^DV increased from 1.12 at pH 7.0 to 1.38 at pH 8.0 and then decreased above pH 8.0. The isotope effect on V_<max> observed at the optimal pH of 9.0 was 1.28. This indicates that the C_3-H bond cleavage step at pH 8.0 is more rate-limiting and that the rate-limiting step changes as a function of pH. However, no significant isotope effect on the C_5-H bond cleavage step was observed in the reaction of L-[5'-^2H]histidine as substrate.

在咪唑环的C-5'处用氘标记的L-组氨酸，L-[5'-^2H]组氨酸，被用作探针以研究组氨酸氨裂解酶（EC 4.3.1.3）催化的氨消除中的酶促反应机制。标记的L-组氨酸与荧光假单胞菌的组氨酸解氨酶在pH7.0或pH9.0、25.0 ℃下孵育24 h。检测反应的时间过程以确定L-组氨酸和尿刊酸的C-5'处的酶催化氢交换速率。酶催化的组氨酸和尿刊酸C-5'位氢交换的发现为消除反应中通过碳负离子中间体的逐步可逆机理提供了合理的解释。在pH7.0时碳负离子的稳定性是pH9.0时的3倍。在pH7.0 -10.5范围内，用L-[3，3-^2H_2]组氨酸和L-[5 '-^2H]组氨酸研究了组氨酸解氨酶反应的氘同位素效应。通过比较标记底物（L-[3，3-^2H_2]组氨酸）和未标记底物的米氏常数，发现<max>V_2和V/K的同位素效应分别为1.00-1.38和1.18-1.63，具有pH依赖性。随着pH的增加，Δ DV从pH 7.0时的1.12增加到pH 8.0时的1.38，然后在pH 8.0以上降低。在<max>最适pH为9.0时，V_2的同位素效应为1.28。这表明在pH8.0时C_3-H键的断裂步骤是一个更强的限速步骤，且限速步骤随pH值的变化而变化，而在以L-[5 '-^2H]组氨酸为底物的反应中，没有观察到明显的同位素效应。