Heterogeneous catalysis by phospholipase A2: formulation of a kinetic description of surface effects

Abstract

The kinetics of hydrolysis of aqueous dispersions of long-chain, saturated phosphatidylcholines (PC) catalyzed by Crotalus atrox venom phospholipase A2 (PLA) were analyzed, and a reaction mechanism proposed which takes surface effects into account. PLA is proposed to form an enzyme-substrate complex with surface substrate molecules, thereby undergoing a conformational change which exposes sites that interact with the lipid surface. After a hydrolytic event, the enzyme can either desorb from the surface (path 1), or diffuse along the surface to an adjacent substrate molecule (path 2). The path 1 dominated mechanism leads to Michaelis-Menten steady-state kinetics, and characterizes hydrolysis of gel phase PC. Evidence for saturation of the surface with PLA was obtained at high enzyme concentrations. The path 2 mechanism dominates when the desorption rate is very small; this mechanism describes hydrolysis of liquid crystalline phase PC and is characterized by an initial burst of hydrolysis followed by a very slow reaction. The velocities in these 2 phases of the reaction are independent of bulk PC concentration. When gel and liquid crystalline PC phases coexist, as in mixtures of dimyristoyl- and distearoyl-PC, the liquid crystalline phase is preferentially hydrolyzed. Products of the reaction (lyso-PC and fatty acid) stimulate hydrolysis, apparently by stimulating desorption of PLA. The desorption rate constant appears to be a linear function of the surface concentrations of lyso-PC and fatty acid. The proposed model describes hydrolysis progress curves extremely well and is consistent with current ideas on the mechanism of catalysis by this enzyme.