Thermodynamic and Kinetic Basis of Interfacial Activation:  Resolution of Binding and Allosteric Effects on Pancreatic Phospholipase A₂ at Zwitterionic Interfaces^,

Abstract

A general kinetic model for catalysis by interfacial enzymes is developed. It couples the Michaelis-Menten catalytic turnover cycle at the interface with that in the aqueous phase through the distribution equilibria between the interface and the surrounding aqueous phase. Analysis under two limiting conditions fully describes the steady-state kinetics of hydrolysis and resolves the allosteric effects from apparent modes of interfacial activation in terms of the primary rate and equilibrium parameters for pig pancreatic phospholipase A2 (PLA2). One limit is observed in dispersions of anionic phospholipid vesicles, in which intervesicle exchange of enzyme, substrate, and hydrolysis products is absent and reaction occurs only on vesicles containing enzyme. A complete analysis at this highly processive limit, called kinetics in the scooting mode, has been published [Berg et al. (1991) Biochemistry 30, 7283]. Here is reported the analysis in the other limit, PLA2-catalyzed hydrolysis of zwitterionic micelles of short-chain phosphatidylcholines, at which substrate and products are in rapid exchange. Hydrolysis occurs either in bulk aqueous solution with phospholipid monomers or at the micellar interface. Above the critical micelle concentration (cmc), the hydrolysis rate shows a hyperbolic dependence on the bulk substrate concentration present as micelles. This dependence, characterized by the fitting parameters KMapp and VMapp, is analyzed in terms of the primary rate and equilibrium constants. The kinetic analysis is based on the assumption that the microscopic steady-state condition is satisfied because substrate replenishment in the micro-environment of the enzyme is fast relative to the catalytic turnover time. Added NaCl and anionic interface increase the hydrolysis rate in zwitterionic micelles dramatically. The overall interfacial rate enhancement is attributed to three factors: (a) promotion of PLA2 binding by net anionic charge of the interface, (b) enhancement of substrate affinity of PLA2 at the interface (Ks* allostery), and (c) stimulation of the rate-limiting chemical step (kcat* allostery).

Crystal Structure of Human Group X Secreted Phospholipase A2

“…Several features of these results are noteworthy especially in relation to the behavior seen with pGIB (41). The rate of hydrolysis below the CMC of DC 7 PC was significant: 20 s Ϫ1 with hGX compared with Ͻ0.03 s Ϫ1 with pGIB.…”

“…Note that V max app remains virtually the same in the presence of 4 M NaCl even though K m app decreases by a factor of three. The decrease in K m app was most likely because of the salt-induced hydrophobic effect, which increases the affinity of the enzyme for the interface and also lowers the CMC of the micellar substrate (41). On the other hand, when comparing rates with 1 mM versus 4 M NaCl added, there was only a marginal change in the value of V max app .…”

“…The initial rate of hydrolysis by hGX of DC 7 PC at several concentrations is shown in Fig. 5, and the kinetic constants, K m app and V max app (41), were extracted from these results and are listed in Table II. These rates, under the stirred or unstirred conditions, were measured by monitoring the fluorescence of a pH indicator dye (28).…”

“…The potential surface is displayed color-coded onto a meshed van der Waals surface using the program GRASP (51), where red and blue represent a net negative and positive charge and white represents overall neutral positions, respectively. a The apparent rate parameters were obtained by fitting the concentration dependence of the observed initial rate under the respective conditions (41). Thus V max app is the initial rate at X s ϭ 1, and K m app is the affinity of the enzyme for the micellar interface.…”

“…However, sPLA 2 enzymes require a bound Ca 2ϩ to bind substrates or substrate mimics. Therefore, according to the detailed balance condition (5,41), the apparent tendency to form a premicellar aggregate would increase in the presence of Ca 2ϩ . The observed formation of such premicellar aggregates makes it quite likely that the hGX catalyzed hydrolysis of substrates proceeds at the interface via an interfacially active E*S form of the enzyme even though the bulk of the substrate amphiphile remains monodisperse in the reaction mixture.…”