Phospholipase A
2 (PLA
2) enzymes become activated by binding to biological membranes andhydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatorymediators. To understand the functional significance of amino acid residues at key positions, we havestudied the effects of the substitution of Val
3 (membrane binding surface) and Phe
5 (substrate bindingpocket) of human group IIA PLA
2 by tryptophan on the structure and function of the enzyme. Despite theclose proximity of the sites of mutations, the V3W mutation results in substantial enhancement of theenzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structuralanalysis of all three proteins free in buffer and bound to membranes indicates that large differences inactivities result from distinct conformational changes in PLA
2s upon membrane binding. Although PLA
2and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, theF5W mutant experiences partial distortion of the
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-helical structure presumably resulting from the tendencyof Trp
5 to insert into the membrane. Furthermore, whereas the PLA
2 and the V3W mutant bind to themembrane at similar and apparently productive-mode orientation, the F5W mutant binds to membraneswith a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutationand the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp
3 at the membrane binding face of PLA
2 facilitates the proper membranebinding of the enzyme, Trp
5 in the internal substrate binding site causes partial unwinding of the
N-terminalhelix in order to interact with the membrane.