Characterization of interfacial catalysis by Aeromonas hydrophila lipase/acyltransferase in the highly processive...

A glycerophospholipid:cholesterol acyltransferase (GCAT) that also has lipase activity is secreted by the bacterium Aeromonas hydrophila. Hydrolysis of the sn-2-ester bond of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) vesicles by this enzyme is shown to occur in a highly processive scooting mode in which the enzyme, substrate, and the products of hydrolysis remain bound to the vesicle interface. This conclusion is based on the following observations. (a) When there is an excess of vesicles over enzyme, the hydrolysis of the sn-2-acyl group ceases after only a fraction of the total available substrate is hydrolyzed. Addition of more enzyme, but not of more substrate, leads to a new round of hydrolysis. (b) The extent of hydrolysis of vesicles per enzyme increases with the size of the vesicles, and it corresponds to the total hydrolysis of the outer monolayer of one vesicle by one enzyme. (c) The enzyme bound to vesicles composed of reaction products or of the non-hydrolyzable phospholipid 1,2-ditetradecyl-sn-glycero-3-phosphomethanol (DTPM) is not able to undergo intervesicle exchange. Instead, intervesicle transfer of the substrate or the bound enzyme due to vesicle fusion promotes hydrolysis of all of the vesicles present in the reaction mixture. (d) Addition of DTPM vesicles to a reaction mixture containing DMPM substrate vesicles and the enzyme has no noticeable effect on the course of hydrolysis. Substrate specificity studies in the scooting mode on DMPM vesicles reveal that GCAT displays essentially no selectivity in the hydrolysis of phospholipids with different polar head groups. Treatment of GCAT with trypsin, which removes a small peptide, results in an enzyme that displays comparable catalytic activity but increased affinity for the interface. Alkyltrifluoromethyl ketones are shown to be tight-binding competitive inhibitors of GCAT. The scooting mode analysis, which has previously been shown to provide a simplified approach for analyzing the steady-state kinetics of interfacial catalysis by secreted phospholipase A2, is also useful for analyzing the interfacial kinetic behavior of lipases.