Kinetic characterization of phospholipase A2 modified by manoalogue.
Kinetic characterization of phospholipase A2 modified by manoalogue.
复制标题
manoalogue 修饰的磷脂酶 A2 的动力学特征。
DOI：
10.1021/bi00104a001
复制
发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Gelb,MH
中科院分区：
文献类型：
--
作者：
Ghomashchi,F;Yu,BZ;Mihelich,ED;Jain,MK;Gelb,MH
Revised Manuscript Received July 8, 1991 abstract: Manoalogue, a synthetic analogue of the sea sponge-derived manoalide, has been previously shown to partially inactivatethe phospholipase A2 from cobra venom (Reynolds, L. J., Morgan, BP, Hite, E. D., Mihelich, E. D., & Dennis, E. A.(1988) J. Am. Chem. Soc. 110, 5172) by reactingwith enzyme lysine residues. In the present study, the inactivation of the phospholipases A2 from pig pancreas, bee venom, and cobra (Naja naja naja) venom by manoalogue was studied in detail. Manoalogue-treated enzymes were examined in the scooting mode on vesicles of l, 2-dimyristoyl-j/z-glycero-3-phosphomethanol. Here, the native enzymes bound irreversibly to the vesicles and hydrolyzedall of the phospholipids in the outer monolayer without leaving the surface of the interface. All three manoalogue-treated enzymes showed reduced catalytic turnover for substrate hydrolysis in thescooting mode, and the modified enzymes did not hop from one vesicle to another. Thus, inactivation by manoalogue is not due to the decrease inthe fraction of enzyme bound to the substrate interface. This result was also confirmed by fluorescence studies that directly monitored the binding of phospolipase A2 to vesicles. A chemically modified form of the pig pancreatic phospholipase A2 in which all of the lysine e-amino groups havebeen amidinated was not inactivated by manoalogue, indicating that the modification of lysine residues and not the amino-terminus is required for the inactivation. Several studies indicated that the manoalogue-modified enzymes contain a functional active site. For example, studies that monitoredthe protection by ligands of the active site from attack by an alkylating agent showed that manoalogue-modified pig phospholipase A2 was capable of binding calcium, a substrate analogue, lipolysis products, and a competitive inhibitor. Furthermore, relative to native enzymes, manoalogue-modified enzymes retained significantly higher catalytic activities when acting on water-soluble substrates than when acting on vesicles in the scooting mode. Intact manoalogue had no affinity for the catalytic site on the enzyme as it did not inhibit the enzyme in the scooting mode and it did not protect the active site from alkylation. Pig pancreatic phospholipase A2 bound to micelles of 2-hexadecyl-i «-glycero-3-phosphocholine was resistant to inactivation by manoalogue, suggesting that the modification of lysine residues on the interfacial recognition surface of the enzyme was required for inactivation. Previous fluorescence studieshave shown that the phospholipid interface in contact with the bound enzymebecomes desolvated and this process may be required for efficient interfacial catalysis inthe scooting mode. In the case of the manoalogue-modified pig phospholipase A2, fluorescence studies indicatedthat the binding of theenzyme to the substrate interface was not accompanied by desolvation of the microinterface in contact with the bound enzyme. Certain properties of manoalogue-modified phospholipase A2 are reminiscent of the proenzyme form of the pig pancreatic enzyme in that they both have significant catalytic activities when acting on soluble substrates and both bind to negatively charged vesicles but in a catalytically nonproductive manner.
