Peters Baron, Trout Bernhardt L
Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Biochemistry. 2006 Apr 25;45(16):5384-92. doi: 10.1021/bi052438n.
Asparagine deamidation is a decisive event in chemotherapy-induced apoptosis and a major obstacle in the formulation of monoclonal antibodies. Despite the importance of deamidation, little is known about the elementary reactions involved. B3LYP/6-31+G(d,p)/COSMO-RS calculations were used to obtain stable structures and transition states for a network of reactions. Calculated rate constants were incorporated into a kinetic model of the pH dependence and compared to a pseudo-steady-state model. At low pH, the calculations show that deamidation occurs by direct acid-catalyzed hydrolysis to aspartate. At neutral to basic pH, deamidation proceeds by the initial formation of a tetrahedral intermediate. The intermediate can be converted to succinimide by two pathways and three rate-determining steps that shift in relative importance with pH. The calculated pH-dependent rate constant qualitatively agrees with the experimental pH dependence. The rate-determining transition state structures may help to understand chemotherapy-induced apoptosis and improve protein formulations.
天冬酰胺脱酰胺作用是化疗诱导细胞凋亡中的一个决定性事件，也是单克隆抗体制剂中的一个主要障碍。尽管脱酰胺作用很重要，但对其中涉及的基本反应却知之甚少。采用B3LYP/6-31+G(d,p)/COSMO-RS计算方法来获得反应网络的稳定结构和过渡态。将计算得到的速率常数纳入pH依赖性动力学模型，并与伪稳态模型进行比较。在低pH值下，计算结果表明脱酰胺作用是通过直接酸催化水解成天冬氨酸而发生的。在中性至碱性pH值下，脱酰胺作用通过首先形成四面体中间体来进行。该中间体可以通过两条途径和三个速率决定步骤转化为琥珀酰亚胺，这些步骤的相对重要性会随pH值而变化。计算得到的pH依赖性速率常数在定性上与实验测得的pH依赖性相符。速率决定过渡态结构可能有助于理解化疗诱导的细胞凋亡并改进蛋白质制剂。
