Calcium-dependent and -independent interfacial binding and catalysis of cytosolic group IV phospholipase A2.

Cytosolic group IV phospholipase A2 (cPLA2) plays a role in liberating arachidonic acid from the sn-2 position of mammalian cellular phospholipids. The enzyme consists of a catalytic domain joined to an N-terminal calcium-dependent, membrane binding domain (C2 domain). The interfacial binding properties of the full-length, nonphosphorylated enzyme and its C2 domain to phospholipid vesicles were studied as a function of vesicle phospholipid composition and calcium concentration. The binding of cPLA2 to phosphatidylcholine vesicles is mostly governed by its C2 domain; binding is relatively weak, and calcium enhances binding and interfacial catalysis by about 10-fold. Catalytically productive interfacial binding was measured by monitoring the increase in the rate of cPLA2-catalyzed hydrolysis of a fluorimetric substrate present in vesicles as a function of bulk vesicle concentration. Enzyme-vesicle binding was also measured by fluorescence as was enzyme-calcium binding. Compared to zwitterionic vesicles, cPLA2 binding to anionic phosphatidylmethanol vesicles is of higher affinity and calcium-independent, although calcium is required for the binding of the C2 domain to these anionic vesicles. cPLA2 is fully catalytically active on phosphatidylmethanol vesicles in the absence of calcium. Phosphatidylserine is not a good replacement for phosphatidylmethanol for inducing high-affinity, calcium-independent binding of cPLA2. These results reveal two modes of catalytically productive interfacial binding of cPLA2: calcium-dependent anchoring via the C2 domain and a calcium-independent component involving a phosphatidylmethanol recognition element in the catalytic domain. They also show that membrane binding of cPLA2 is not, in general, predicted by the interfacial binding properties of its C2 domain.