INTERFACIAL ENZYMOLOGY

The primary focus of my research efforts is interfacial catalysis and activation with a focus on phospholipase A₂. With this paradigm, we address key issues of interfacial enzymology, including kinetics in two-dimension, lipid-protein interactions, phospholipid exchange mediated by proteins.

A general, quantitative and analytical description of interfacial catalysis in terms of the primary rate and equilibrium constants is shown in the kinetic Scheme. As an extension of the Michaelis-Menten formalism, the Scheme is elegantly simple yet it is remarkably versatile. The overall catalytic turnover for interfacial enzymology is a two step process: binding of the enzyme from the aqueous phase to the interface, and the catalytic turnover on the interface. Catalytic turnover through the monomer path is negligible, i.e. k*_cat/k_cat > 10⁵ for several secreted PLA2. Even with this simplification, as such there are far too many constants and variable intrinsic in this kinetic scheme due to the exchange of the reactants between the two compartments. By constraining certain variables under three limiting conditions, we have completely analyzed the steady-state interfacial kinetics in terms of the primary rate and equilibrium constants:

(a) In the scooting mode, the enzyme exchange between vesicles is constrained to the pre-steady state. Also the exchange of natural phospholipid substrates and products is very slow on the kinetic time-scale. This permits dissection of the steps, shown in the oval, for the interfacial kinetic turnover.

(b) With rapidly exchanging substrate and products, the interfacial chemical step can remain rate limiting, as is the case with micelles of short-chain phospholipid. Since this condition is not satisfied with mixed-micelles of natural phospholipid with detergents, where the substrate replenishment in the enzyme containing micelles becomes rate-limiting.

(c) Rapidly exchanging substrate and products partitioned in a diluent interface, to which the enzyme binds, is also amenable to the analysis.

Having established the theory and experimental protocols to unequivocally resolve the primary interfacial kinetic and equilibrium parameters under the three limiting conditions, we have developed detailed quantitative understanding of the variables that control the observed kinetics at interfaces. Some of the areas which we are pursuing and others which we plan to follow up in the near future are outlined below:

1. Specific inhibitors of secreted phospholipase A₂. In the past there was no known method to unequivocally assay inhibitors of PLA. Assays based on scooting kinetics identify specific competitive inhibitors without other complications. This has yielded several inhibitors of pharmacological and physiological interest, and provided useful mechanistic information.
2. Interfacial kinetic rate constants (k₁, k_-1, k₂ etc.) for the catalytic turnover contain information about the primary mechanism of interfacial activation. The substrate concentration that the enzyme "sees" in the interface is not the bulk concentration, but the number density (approximated as the mole fraction in the interface). Thus the dissociation constants in the interface (inhibitors, products, or substrate analogous) are expressed in mole fraction units.
3. Site directed mutagenesis to identify role of residues in catalytic function (substrate and calcium binding), and interfacial activation and interfacial recognition.

Overall, our research efforts are problem-oriented with a view towards understanding the fundamental molecular and biochemical processes at lipid-water interfaces in as much detail as possible. In this sense PLA is not only a prototype for interfacial catalysis in general but it could be a reasonable model for certain types of high affinity lipid-protein interactions. In a way, the most exciting period of our research efforts has just begun, where we can apply now-established techniques and protocols will provide complete understanding of interfacial enzymology.