Surface dilution kinetics of phospholipase A(2) catalyzed lipid-bilayer hydrolysis.
Phospholipase A2 (PLA2) enzymes catalyze hydrolysis of phospholipids in membranes. Elucidation of the kinetics of interfacial enzymatic activity is best accomplished by investigating the interface substrate concentration dependence of the activity for which appropriate diluents are required. PLA2 is stereoselective toward the L_enantiomers of phospholipids. A novel approach employing D_phospholipids as diluents to perform surface dilution kinetic studies of PLA2 is presented. Activity of bee venom PLA2 at mixed L+D_DPPC (dipalmitoylphosphatidylcholine) bilayer interfaces was measured as a function of substrate L_DPPC mole fraction and vesicle concentration using a sensitive fluorescence assay. A model for interface enzymatic activity based on the three-step kinetic scheme of (i) binding of PLA2 to the bilayer interface, (ii) binding of a lipid to PLA2 at the interface, and (iii) hydrolysis was applied to the hydrolysis data. Activity profiles showed that D_enantiomers also bind to the enzyme but resist hydrolysis. Activity dependences on vesicle and substrate concentrations could be disentangled, bringing resolution to an outstanding problem in membrane hydrolysis of separating the effects of the three steps. Individual values of the kinetic parameters of the model, including the vesicle-PLA2 equilibrium dissociation constant of step (i), interface Michaelis-Menten-Henri constant for L and D_DPPC of step (ii), and the rate constant for interface hydrolysis, step (iii), were obtained as solutions to equations resulting from fitting the model to the data.
Department of Physics, California State University Northridge and Center for Supramolecular Studies , Northridge, California 91330-8268, United States.
Jasmeet
J
Singh
Radha
R
Ranganathan
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry / Q000378:metabolism; D001514:Bee Venoms / Q000201:enzymology; D055162:Biocatalysis; D006868:Hydrolysis; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D054467:Phospholipases A2 / Q000378:metabolism*; D013237:Stereoisomerism; D013499:Surface Properties
D001514:Bee Venoms; D008051:Lipid Bilayers; D015060:1,2-Dipalmitoylphosphatidylcholine; D054467:Phospholipases A2