Changes in Ca2+ affinity upon activation of Agkistrodon piscivorus piscivorus phospholipase A2.
Changes in the affinity of calcium for phospholipase A2 from Agkistrodon piscivorus piscivorus during activation of the enzyme on the surface of phosphatidylcholine vesicles have been investigated by site-directed mutagenesis 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 generating a panel of mutant proteins, each of which lacks one or more Trp residues. Both Trp21, which is found in the interfacial binding region, and Trp119 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 fluorescence of the protein also occurs during activation of the enzyme. These changes arise from residue Trp31 as well as residues Trp21 and Trp119. The calcium dependence of the fluorescence change of Trp31 indicates that the affinity 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.
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
B
B
Lathrop
M
M
Gadd
R L
RL
Biltonen
G S
GS
Rule
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D017836:Agkistrodon; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D003435:Crotalid Venoms / Q000201:enzymology*; D004789:Enzyme Activation / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D050356:Lipid Metabolism; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
D003435:Crotalid Venoms; D008051:Lipid Bilayers; D011994:Recombinant Proteins; D012996:Solutions; D015060:1,2-Dipalmitoylphosphatidylcholine; D014364:Tryptophan; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium