Kinetics of binding of phospholipase A2 to lipid/water interfaces and its relationship to interfacial activation.
The time-course of binding of phospholipase A2 and prophospholipase A2 to vesicles and micelles of a variety of substrate and nonhydrolyzable phospholipid analogs is obtained by monitoring the change in the fluorescence intensity of Trp-3 on the protein or of the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) chromophore on the surface of the vesicles. The time-dependent increase in the fluorescence intensity of phospholipase A2 is observed only under conditions where catalysis and equilibrium binding are also observed. The overall kinetics of binding is described by two rate constants. A rapid second-order rate constant (ka) for binding of both the proteins is 2.10(7) per s per mol expressed in terms of phospholipids as monomers, and 10(10) per s per mol expressed in terms of vesicles. This is probably a diffusion-limited encounter of the protein with vesicles as the first step in binding. An additional first-order rate constant (kb = 4 per s) was also discerned for the binding of phospholipase A2 but not for prophospholipase A2. The rate of desorption of the bound iphospholipase A2 in the presence of EGTA is very slow (less than 0.0002 per s), whereas the rate of desorption of the bound prophospholipase A2 is much more rapid (2.9 per s). The mechanistic significance of these rate constants is elaborated in terms of the differences in the rates of interfacial catalytic turnover of phospholipase A2 and prophospholipase A2. As shown elsewhere (Jain et al. Biochim. Biophys. Acta 860, 435-447) the hydrolysis of anionic vesicles by phospholipase A2 occurs in the scooting mode such that the bound enzyme remains on the target vesicles for several thousand catalytic turnover cycles. On the other hand, as shown in this paper, the kinetics of hydrolysis by prophospholipase A2 is dominated by its intervesicle exchange. Therefore, interfacial catalysis by prophospholipase A2 in the hopping mode would involve an on- and an off-step in each cycle, resulting in a catalytic turnover number of about 1.2 per s. A change from the hopping to the scooting mode of catalysis thus provides the kinetic basis for activation of interfacial catalysis by phospholipase A2 compared to that for prophospholipase A2.
Jain
M K
MK
Department of Chemistry, University of Delaware, Newark 19716.
Rogers
J
J
DeHaas
G H
GH
D000818:Animals; D004789:Enzyme Activation; D007700:Kinetics; D008823:Micelles; D008962:Models, Theoretical; D010179:Pancreas / Q000201:enzymology; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids; D013050:Spectrometry, Fluorescence; D013499:Surface Properties; D013552:Swine; D013816:Thermodynamics; D014867:Water
D008823:Micelles; D010743:Phospholipids; D014867:Water; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2