B. Lathrop et al., Changes in Ca2+ affinity upon activation of Agkistrodon piscivorus piscivorus phospholipase A(2), BIOCHEM, 40(11), 2001, pp. 3264-3272
Changes in the affinity of calcium for phospholipase A(2) from Agkistrodon
piscivorus piscivorus during activation of the enzyme on the surface of pho
sphatidylcholine vesicles have been investigated by site-directed mutagenes
is and fluorescence spectroscopy. Changes in fluorescence that occur during
lipid binding and subsequent activation have been ascribed to each of the
three individual Trp residues in the protein, This was accomplished by gene
rating a panel of mutant proteins, each of which lacks one or more Trp resi
dues. Both Trp21, which is found in the interfacial binding region, and Trp
119 show changes in fluorescence upon protein binding to small unilamellar
zwitterionic vesicles or large unilamellar vesicles containing sufficient
anionic lipid. Trp31, which is near the Ca2+ binding loop, exhibits little
change in fluorescence upon lipid bilayer binding. A change in the fluoresc
ence of the protein also occurs during activation of the enzyme. These chan
ges arise from residue Trp31 as well as residues Trp21 and Trp119, The calc
ium dependence of the fluorescence change of Trp31 indicates that the affin
ity of the enzyme for calcium increases at least 3 orders of magnitude upon
activation. These studies suggest either that a change in conformation of
the enzyme occurs upon activation or that the increase in calcium affinity
reflects formation of a ternary complex of calcium, enzyme, and substrate.