Bayburt T, Yu B Z, Lin H K, Browning J, Jain M K, Gelb M H
Department of Chemistry, University of Washington, Seattle 98195.
Biochemistry. 1993 Jan 19;32(2):573-82. doi: 10.1021/bi00053a024.
The rate and equilibrium parameters for the interfacial catalysis by recombinant human nonpancreatic secreted phospholipase A2 were determined. Results show that the enzyme binds to anionic interfaces with considerably higher affinity than to zwitterionic interfaces. The extent of hydrolysis per enzyme on anionic vesicles in the processive scooting mode shows that the enzyme is fully catalytically active as a monomer. Among several secreted phospholipases A2 tested, the human nonpancreatic secreted enzyme is unique in its ability to undergo slow intervesicle exchange either by dissociation from the interface followed by binding to a different vesicle or by promoting the fusion of vesicles. The equilibrium dissociation constants for calcium, substrate analogs, reaction products, and several competitive inhibitors bound to the enzyme at the interface were determined by monitoring the ligand-conferred protection of the active site histidine residue from alkylation by phenacyl bromide. The interfacial Michaelis-Menten parameters were determined from the analysis of the entire reaction progress curve and also by monitoring the effect of competitive inhibitors on the initial rate of hydrolysis in the scooting mode. The interfacial Michaelis constant (KM*) for the substrate 1,2-dimyristoylglycero-sn-3-phosphomethanol was determined to be considerably above the maximal attainable mole fraction of unity for the substrate in the bilayer. Substrate specificity studies show that the enzyme does not significantly discriminate between phospholipids that differ in the type of polar head group or in the degree of unsaturation of the fatty acyl chains. Competitive inhibitors are described that display a high degree of selectivity for binding to the nonpancreatic versus pancreatic phospholipase A2. The kinetic properties of the human nonpancreatic secreted phospholipase A2 suggest that the enzyme has evolved to hydrolyze substrates at anionic interfaces and at high calcium concentrations.
测定了重组人非胰腺分泌型磷脂酶A2的界面催化速率和平衡参数。结果表明，该酶与阴离子界面的结合亲和力远高于两性离子界面。在连续滑动模式下，该酶在阴离子囊泡上的水解程度表明其作为单体具有完全的催化活性。在几种测试的分泌型磷脂酶A2中，人非胰腺分泌型酶具有独特的能力，能够通过从界面解离后结合到不同的囊泡或促进囊泡融合来进行缓慢的囊泡间交换。通过监测配体对活性位点组氨酸残基免受苯甲酰溴烷基化的保护作用，测定了与界面处酶结合的钙、底物类似物、反应产物和几种竞争性抑制剂的平衡解离常数。界面米氏参数通过对整个反应进程曲线的分析以及监测竞争性抑制剂对滑动模式下水解初始速率的影响来确定。底物1,2 - 二肉豆蔻酰甘油 - sn - 3 - 磷酸甲醇的界面米氏常数（KM*）被确定为远高于双层中底物最大可达到的摩尔分数1。底物特异性研究表明，该酶对极性头部基团类型或脂肪酰链不饱和度不同的磷脂没有明显区分。描述了对非胰腺型与胰腺型磷脂酶A2具有高度选择性结合的竞争性抑制剂。人非胰腺分泌型磷脂酶A2的动力学性质表明，该酶已进化为在阴离子界面和高钙浓度下催化底物水解。
