Kinetic characterization of phospholipase A2 modified by manoalogue.
Manoalogue, a synthetic analogue of the sea sponge-derived manoalide, has been previously shown to partially inactivate the phospholipase A2 from cobra venom (Reynolds, L. J., Morgan, B. P., Hite, E. D., Mihelich, E. D., & Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172) by reacting with 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 1,2-dimyristoyl-sn-glycero-3-phosphomethanol. Here the native enzymes bound irreversibly to the vesicles and hydrolyzed all 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 the scooting mode, and the modified enzymes did not hop from one vesicle to another. Thus, inactivation by manoalogue is not due to the decrease in the fraction of enzyme bound to the substrate interface. This result was also confirmed by fluorescence studies that directly monitored the binding of phospholipase A2 to vesicles. A chemically modified form of the pig pancreatic phospholipase A2 in which all of the lysine epsilon-amino groups have been 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 monitored the protection by ligands of the active site from attack by a 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-sn-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.(ABSTRACT TRUNCATED AT 400 WORDS)
Ghomashchi
F
F
Department of Chemistry, University of Washington, Seattle 98195.
Yu
B Z
BZ
Mihelich
E D
ED
Jain
M K
MK
Gelb
M H
MH
D000818:Animals; D001667:Binding, Competitive; D002384:Catalysis; D004789:Enzyme Activation; D020404:Glycerophospholipids; D007700:Kinetics; D008823:Micelles; D010712:Phosphatidic Acids; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000187:drug effects; D054467:Phospholipases A2; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine; D013729:Terpenes / Q000138:chemical synthesis / Q000494:pharmacology*
D020404:Glycerophospholipids; D008823:Micelles; D010712:Phosphatidic Acids; D013729:Terpenes; C070695:manoalogue; C029313:dimyristoylmethylphosphatidic acid; D010741:Phospholipases A; D054467:Phospholipases A2