INTERFACIAL CATALYSIS BY PHOSPHOLIPASE-A2 - DETERMINATION OF THE INTERFACIAL KINETIC RATE CONSTANTS
INTERFACIAL CATALYSIS BY PHOSPHOLIPASE-A2 - DETERMINATION OF THE INTERFACIAL KINETIC RATE CONSTANTS
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DOI：
10.1021/bi00243a034
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发表时间：
1991-07-23
期刊：
影响因子：
2.9
通讯作者：
JAIN, MK
中科院分区：
文献类型：
--
作者：
BERG, OG;YU, BZ;JAIN, MK
猪胰腺磷脂酶A2 （PLA2）水解1,2-二myristoyl- asn -glycero-3- phosphome（DMPM）囊泡的过程是高度“快速”的，其速率与在最佳条件下用洗涤剂分散的混合胶束所观察到的速率相当或超过。建立了水解稳态时间过程的完整动力学描述。该分析涵盖了整个Michaelis-Menten空间：它通过详细的理论分析强调了界面催化的关键特征，描述了确定界面催化动力学和平衡常数值的实验方案，并通过一组速率和平衡参数解释了钙、底物、产物、表观活化剂和竞争抑制剂对反应过程曲线的影响。本文根据界面上Michaelis-Menten反应顺序（E* + S可逆E*S—> E*P可逆E* + P）的最小模型对反应过程曲线进行了严格的解释，并确定了催化循环的大部分单个速率和平衡常数。这种对界面催化的严格描述是通过实验来实现的，在每个囊泡最多有一个酶的条件下，PLA2在滑板模式下的作用是可能的，在这种情况下，它水解了囊泡外单层中的所有底物而不离开表面。该分析的其他实验验证约束包括：所有酶都与囊泡结合；在水解过程中保持了囊泡的完整性；底物、酶和产物不会在囊泡之间交换，也不会在双分子层之间交换。附文讨论了速率常数的力学意义。
Hydrolysis of vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) by pig pancreatic phospholipase A2 (PLA2) occurs in a highly processive "scooting" mode, and the rate is comparable to or exceeds the rates observed with detergent-dispersed mixed micelles under optimal conditions. A complete kinetic description of the steady-state time course of the hydrolysis is developed. The analysis covers the whole Michaelis-Menten space: it emphasizes the key features of interfacial catalysis by a detailed theoretical analysis, describes the experimental protocols to determine the values of the kinetic and equilibrium constants for interfacial catalysis, and provides an interpretation of the effect of calcium, substrate, products, apparent activators, and competitive inhibitors on the reaction progress curve by a single set of rate and equilibrium parameters. In this paper, the integrated reaction progress curve was rigorously interpreted in terms of a minimal model involving the Michaelis-Menten reaction sequence in the interface: E* + S reversible E*S --> E*P reversible E* + P, and most of the individual rate and equilibrium constants for the catalytic cycle were determined. This rigorous description of interfacial catalysis was made experimentally possible by examining the action of PLA2 in the scooting mode under conditions of at most one enzyme per vesicle, where it hydrolyzed all of the substrate in the outer monolayer of vesicles without leaving the surface. Other experimentally verified constraints for this analysis include the following: all enzyme was bound to vesicles; the integrity of vesicles was maintained during the course of hydrolysis; and the substrate, enzyme, and products did not exchange between vesicles nor did they exchange across the bilayer. The mechanistic significance of the rate constants is discussed in the accompanying papers.