A strategy is developed to analyze steady-state kinetics for the hydrolysis of a soluble substrate partitioned into the interface by an enzyme at the interface. The feasibility of this approach to obtain interfacial primary kinetic and equilibrium parameters is demonstrated for a triglyceride lipase. Analysis for phospholipase A2 catalyzed hydrolysis of rapidly exchanging micellar (Berg et al. (1997) Biochemistry 36, 14512-14530) and nonexchangeable vesicular (Berg et al., (1991) Biochemistry 30, 7283-7291) phospholipids is extended to include the case of a substrate that does not form the interface. The triglyceride lipase (tlTGL) from Thermomyces (formerly Humicola) lanuginosa hydrolyzes p-nitrophenylbutyrate or tributyrin partitioned in the interface of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoglycerol (POPG) vesicles at a rate that is more than 100-fold higher than that for the monodispersed substrate or for the substrate partitioned into zwitterionic vesicles. Catalysis and activation is not seen with the S 146A mutant without the catalytic serine-146; however, it binds to the POPG interface with the same affinity as the WT. Thus POPG acts as a diluent surface to which the lipase binds in an active, or 'open', form for the catalytic turnover; however, the diluent molecules have poor affinity for the active site. Analysis of the substrate and the diluent concentration dependence of the rate of hydrolysis provides a basis for the determination of the primary interfacial catalytic parameters. As a competitive substrate, tributyrin provided a check for the apparent affinity parameters. Nonidealities from the fractional difference in the molecular areas in interfaces are expressed as the area correction factor and can be interpreted as a first-order approximation for the interfacial activity coefficient. The basis for the interfacial activation of tlTGL on anionic interface is attributed to cationic R81, R84, and K98 in the 'hinge' around the 86-93 'lid' segment of tlTGL.
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