TRYPTOPHAN-CONTAINING MUTANT OF HUMAN (GROUP IIA) SECRETED PHOSPHOLIPASE A(2) HAS A DRAMATICALLY INCREASED ABILITY TO HYDROLYZE PHOSPHATIDYLCHOLINE VESICLES AND CELL-MEMBRANES

Human nonpancreatic (group IIa) secreted phospholipase A(2) (human sPL A(2)) is associated with a number of inflammatory disorders in which t he extracellular concentrations of this enzyme can become highly eleva ted. It is probable that the enzyme normally acts as an acute-phase pr otein whose function is to facilitate the removal of infectious organi sms or damaged host cells as part of the normal inflammatory response. The enzyme shows negligible activity with phosphatidylcholine (PC) ve sicles and cell membranes, presumably reflecting the enzyme's lack of ability to bind productively to such condensed neutral interfaces. Mam malian pancreatic enzymes show modest activity with such interfaces an d contain a unique tryptophan at position 3, which is part of the pres umptive interfacial binding: surface of these enzymes. Human sPLA(2) d oes not contain tryptophan. The amphiphilic indole side chain of trypt ophan is noted for its ability to penetrate the lipid interface of mem branes, and tryptophan residues appear to be associated with the abili ty of lipases and phospholipases A(2) to bind to and hydrolyze such in terfaces. We have investigated in detail the properties of a V3W mutan t of human sPLA(2), which has a unique tryptophan on the interfacial b inding surface of this enzyme. Although this enzyme shows a modest (si milar to 50%) reduction in activity when anionic substrates are used u nder standard assay conditions, the activity of the enzyme on phosphat idylcholine vesicles and cell membranes is dramatically increased comp ared with human sPLA(2). This is particularly the case with small unil amellar vesicles of PC, where activity is enhanced over 250-fold compa red to the almost zero activity expressed by human sPLA(2), This enhan ced activity is best explained by increased interfacial binding and ac tivation of the V3W mutant and is not due to enhanced active-site bind ing and hydrolysis. The results highlight the important role that tryp tophan residues can play in interfacial binding, particularly to conde nsed zwitterionic interfaces, The interfacial characteristics of the m utant human enzyme now resemble more closely the mammalian pancreatic enzymes that already have a tryptophan at position 3.