Thermodynamic and kinetic basis of interfacial activation: resolution of binding and allosteric effects on pancreatic phospholipase A2 at zwitterionic interfaces.
Thermodynamic and kinetic basis of interfacial activation: resolution of binding and allosteric effects on pancreatic phospholipase A2 at zwitterionic interfaces.
复制标题
界面激活的热力学和动力学基础：两性离子界面上胰腺磷脂酶 A2 的结合和变构效应的解析。
作者：
Berg,OG;Rogers,J;Yu,BZ;Yao,J;Romsted,LS;Jain,MK
建立了界面酶催化的一般动力学模型。它通过界面与周围水相之间的分布平衡将界面上的Michaelis - Menten催化周转循环与水相中的周转循环耦合在一起。在两个极限条件下的分析充分描述了水解的稳态动力学，并根据猪胰磷脂酶A2（PLA2）的主要速率和平衡参数，解决了界面激活的表观模式的变抗效应。在阴离子磷脂囊泡的分散中观察到一个限制，其中酶、底物和水解产物在囊泡间没有交换，反应只发生在含有酶的囊泡上。对这种被称为踏板车模式动力学的高度过程极限的完整分析已经发表[Berg et al. (1991) biochemistry, 30,7283]。本文报道了pla2催化短链磷脂酰胆碱两性离子胶束水解的另一极限分析，在该极限下，底物和产物进行了快速交换。水解既发生在含有磷脂单体的散装水溶液中，也发生在胶束界面上。在临界胶束浓度（cmc）以上，水解速率与作为胶束存在的体积底物浓度呈双曲线关系。以拟合参数skmappand vmapp为特征的这种依赖关系是根据初级速率和平衡常数进行分析的。动力学分析是基于满足微观稳态条件的假设，因为底物在酶的微环境中补充相对于催化周转时间快。加入NaCl和阴离子界面可显著提高两性离子胶束的水解速率。整体界面速率的提高归因于三个因素：(a)界面净阴离子电荷促进PLA2结合，(b)增强PLA2在界面的底物亲和力（KS*变构），以及(c)刺激限速化学步骤（kcat*变构）。
A general kinetic model for catalysis by interfacial enzymes is developed. It couples the Michaelis−Menten catalytic turnover cycle at the interface with that in the aqueous phase through the distribution equilibria between the interface and the surrounding aqueous phase. Analysis under two limiting conditions fully describes the steady-state kinetics of hydrolysis and resolves the allosteric effects from apparent modes of interfacial activation in terms of the primary rate and equilibrium parameters for pig pancreatic phospholipase A2(PLA2). One limit is observed in dispersions of anionic phospholipid vesicles, in which intervesicle exchange of enzyme, substrate, and hydrolysis products is absent and reaction occurs only on vesicles containing enzyme. A complete analysis at this highly processive limit, called kinetics in the scooting mode, has been published [Berg et al. (1991)Biochemistry30, 7283]. Here is reported the analysis in the other limit, PLA2-catalyzed hydrolysis of zwitterionic micelles of short-chain phosphatidylcholines, at which substrate and products are in rapid exchange. Hydrolysis occurs either in bulk aqueous solution with phospholipid monomers or at the micellar interface. Above the critical micelle concentration (cmc), the hydrolysis rate shows a hyperbolic dependence on the bulk substrate concentration present as micelles. This dependence, characterized by the fitting parametersKMappandVMapp, is analyzed in terms of the primary rate and equilibrium constants. The kinetic analysis is based on the assumption that the microscopic steady-state condition is satisfied because substrate replenishment in the microenvironment of the enzyme is fast relative to the catalytic turnover time. Added NaCl and anionic interface increase the hydrolysis rate in zwitterionic micelles dramatically. The overall interfacial rate enhancement is attributed to three factors: (a) promotion of PLA2 binding by net anionic charge of the interface, (b) enhancement of substrate affinity of PLA2 at the interface (KS* allostery), and (c) stimulation of the rate-limiting chemical step (kcat* allostery).