title: Interfacial catalysis by phospholipase A2: evaluation of the interfacial rate constants by steady-state isotope effect studies.
abstract: The kinetics of hydrolysis of phospholipid vesicles by phospholipase A2 (PLA2) in the scooting mode can be described by the Michaelis-Menten formalism for the action of the enzyme in the interface (E*). E* + S in equilibrium E*S in equilibrium E*P in equilibrium E* + Products The values of the interfacial rate constants cannot be obtained by classical methods because the concentration of the substrate within the lipid bilayer is not easily manipulated. In the present study, carbonyl-carbon heavy atom isotope effects for the hydrolysis of phospholipids have been measured in both vesicles and in mixed micelles in which the phospholipid was present in the nonionic detergent Triton X-100. A large [14C]carbonyl carbon isotope effect of 1.12 +/- 0.02 was measured for the cobra venom PLA2-catalyzed hydrolysis of dipalmitoylphosphatidylcholine in Triton X-100. In contrast, no isotope effect (1.01 +/- 0.01) was measured for the action of the porcine pancreatic and cobra venom enzymes on vesicles of dimyristoylphosphatidylmethanol in the scooting mode. In a second experiment, the hydrolysis of vesicles was carried out in oxygen-18 enriched water. Analysis of the released fatty acid product by mass spectrometry showed that it contained only a single oxygen-18. All of these results were used to estimate both the forward and reverse commitments to catalysis. The lack of doubly labeled fatty acid demonstrated that the product is released from the E*P complex faster than the reverse of the esterolysis step. The small isotope effect in vesicles demonstrated that the E*S complex goes on to products faster than substrate is released from the enzyme. The relevance of these results to an understanding of substrate specificity and inhibition of PLA2 is discussed. In addition, the conditions placed on the values of the rate constants obtained in the present study together with results obtained in the other studies described in this series of papers have led to the evaluation of most of the interfacial rate constants for the hydrolysis of phospholipid vesicles by PLA2.
authors.affiliation: Department of Chemistry, University of Washington, Seattle 98195.
authors.forename: F
authors.initials: F
authors.lastname: Ghomashchi
authors.forename: T
authors.initials: T
authors.lastname: O'Hare
authors.forename: D
authors.initials: D
authors.lastname: Clary
authors.forename: M H
authors.initials: MH
authors.lastname: Gelb
mesh_terms: D000818:Animals; D002384:Catalysis; D004546:Elapid Venoms; D020404:Glycerophospholipids; D006868:Hydrolysis; D007553:Isotope Labeling; D007700:Kinetics; D008823:Micelles; D010103:Oxygen Isotopes; D010179:Pancreas / Q000201:enzymology; D010712:Phosphatidic Acids / Q000737:chemistry; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D013379:Substrate Specificity; D013552:Swine
chemical_list: D004546:Elapid Venoms; D020404:Glycerophospholipids; D008823:Micelles; D010103:Oxygen Isotopes; D010712:Phosphatidic Acids; C029313:dimyristoylmethylphosphatidic acid; D010741:Phospholipases A; D054467:Phospholipases A2