Physical-chemical requirements for the catalysis of substrates by lysosomal phospholipase A1.

The catalytic properties of a 1440-fold purified preparation of lysosomal phospholipase A1 were examined. The preparation was at least 95% specific for the sn-1 position of neat phosphatidylethanolamine (PE). The apparent specificity of the enzyme toward substrates was affected by three factors: the physical arrangement of molecules in the substrate aggregate, the charge on the lipid-water interface and the chemical structure of the substrate as it relates to the active site of the enzyme. Of various phospholipids tested in the absence of detergent PE was the preferred substrate, phosphatidylcholine (PC) was hydrolyzed at one-fifth the rate of PE, while phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylglycerol (PG) were degraded very slowly. Triton WR1339 stimulated the hydrolysis of PC, PI, PS, and PG but inhibited the hydrolysis of PE, with PG the preferred substrate at a 6:1 Triton/phospholipid ratio. The preference for PC over PE in detergent mixtures was attributed to the active site fit of the chemical structures of the substrate molecules. The enzyme preferentially hydrolyzed neat PE containing palmitic and oleic acids at position 1. A negative surface charge was required for the hydrolysis of PC and PE. Ca2+ stimulated the hydrolysis of PI, PS, and PG but inhibited the hydrolysis of PE. The inhibition of PE hydrolysis by Ca2+ was the result of an alteration in the surface charge of the PE vesicle. Chromatography of phospholipase A1 on concanavalin A-Sepharose resulted in a loss of activity toward acidic phospholipids which could be restored with Ca2+. Plasmalogen PE was found to inhibit the hydrolysis of diacyl-PE at the level of interfacial binding but not by competition for the active site of the enzyme. These results suggest that the hexagonal structure of PE represents a preferred physical form for catalysis by phospholipase A1, while the bilayer form is less readily attacked. Dispersion of the substrate in the inert detergent enhanced the activity of those substrates normally forming bilayer structures. We demonstrate the importance of the "quality of interface" in regulating the activity of the enzyme.