Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry.
Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry.
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通过结构相关的动力学和计算技术确定木瓜蛋白酶和番薯碱的天冬氨酸残基 158 的电离特性和化学影响：活性中心化学的多个静电调制器。
DOI：
10.1042/bj3510723
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
K. Brocklehurst
中科院分区：
文献类型：
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作者：
Michael A. Noble;Sheraz Gul;Chandra S. Verma;K. Brocklehurst
以4-氯-7-硝基苯并呋咱（Nbf-Cl）为探针，以催化位点的Cys（25）/His（159）亲核酸/碱基序的硫醇阴离子组分为靶点，测定木瓜蛋白酶（EC3.4.22.2）的（Asp（158））-CO（2）H的pK（a）为2.8。通过使用QUANTA/CHARMM对木瓜蛋白酶-Nbf-Cl Meisenheimer中间体进行建模，并使用与Cerius 2接口的MOPAC进行分子轨道计算，确定了使用Nbf-Cl用于此目的的可能性。木瓜蛋白酶与Nbf-Cl反应的pH依赖性停流动力学研究表明，pH 3时的最大撞击速率是由包含（Cys（25））-S（-）/（His（159））-Im（+）H的次要电离态的反应引起的（其中Im代表咪唑）是由pK（a）3.3的（Cys（25））-SH/（His（159））-Im（+）H和（Asp（158））-CO（2）H的质子解离产生的。尽管在洋甘菊蛋白酶（EC 3.4.22.30）与Nbf-Cl的反应中类似的中间体具有相似的几何形状，但pH-k曲线（k为二级速率常数）在酸性条件下缺乏速率最大值。这就排除了实验测定洋甘菊蛋白酶的（Asp（158））-CO（2）H的pK（α）值的可能性，通过用程序UHBD（'University of Houston Brownian dynamics'）求解线性化的Poisson-Boltzmann方程，计算出pK（α）值为2.0。通过考虑两种酶中涉及Asp（158）及其微环境的氢键网络，预测了低于2.8的值。这些pK（a）值之间的差异（先前在任一酶的反应中未检测到的值）解释了在胭脂虫酶反应中缺乏速率最大值以及在酸性条件下两种S-Nbf-酶的电子吸收光谱的差异。本文讨论了多种静电调节剂（Asp（158））-CO（2）（-）对半胱氨酸蛋白酶活性的调控，改变了传统的机理观点。
The pK(a) of (Asp(158))-CO(2)H of papain (EC 3.4.22.2) was determined as 2.8 by using 4-chloro-7-nitrobenzofurazan (Nbf-Cl) as a reactivity probe targeted on the thiolate anion component of the Cys(25)/His(159) nucleophilic-acid/base motif of the catalytic site. The possibility of using Nbf-Cl for this purpose was established by modelling the papain-Nbf-Cl Meisenheimer intermediate by using QUANTA/CHARMM and performing molecular orbital calculations with MOPAC interfaced with Cerius 2. A pH-dependent stopped-flow kinetic study of the reaction of papain with Nbf-Cl established that the striking rate maximum at pH 3 results from reaction in a minor ionization state comprising (Cys(25))-S(-)/(His(159))-Im(+)H (in which Im represents imidazole) produced by protonic dissociation of (Cys(25))-SH/(His(159))-Im(+)H with pK(a) 3.3 and (Asp(158))-CO(2)H. Although the analogous intermediate in the reaction of caricain (EC 3.4.22.30) with Nbf-Cl has similar geometry, the pH-k profile (k being the second-order rate constant) lacks a rate maximum under acidic conditions. This precludes the experimental determination of the pK(a) value of (Asp(158))-CO(2)H of caricain, which was calculated to be 2.0 by solving the linearized Poisson-Boltzmann equation with the program UHBD ('University of Houston Brownian dynamics'). A value lower than 2.8 had been predicted by consideration of the hydrogen-bonded networks involving Asp(158) and its microenvironments in both enzymes. The difference between these pK(a) values (values not previously detected in reactions of either enzyme) accounts for the lack of the rate maximum in the caricain reaction and for the differences in the electronic absorption spectra of the two S-Nbf-enzymes under acidic conditions. The concept of control of cysteine proteinase activity by multiple electrostatic modulators, including (Asp(158))-CO(2)(-), which modifies traditional mechanistic views, is discussed.