THE ESSENTIALITY OF CALCIUM-ION IN THE ENZYMATIC-ACTIVITY OF TAIWAN COBRA PHOSPHOLIPASE A(2)

In order to address the mechanism whereby Ca2+ wad crucial for the man ifestation of the enzymatic activity of phospholipase A(2) (PLA(2)), f our divalent cations were used to assess their influences on the catal ytic activity and the fine structures of Naja naja atra PLA(2). It was found that substitution of Mg2+ or Sr2+ for Ca2+ in the substrate sol ution caused a decrease in the PLA(2) activity to 77.5% or 54.5%, resp ectively, of that in the presence of Ca2+. However, no PLA(2) activity was observed with the addition of Ba2+. With the exception of Mg2+, t he nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding s ite of PLA(2) markedly increased with the binding of cations to PLA(2) . In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) to ward trinitrobenzene sulfonate and p-nitrobenzenesulfonyl fluoride wer e enhanced by the addition of Ca2+, Sr2+, and Ba2+, but not by Mg2+. T he order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was B a2+ > Sr2+ > Ca2+ > Mg2+, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA(2) and that Tyr-3, L ys-6, and Tyr-63 of PLA(2) are involved in the binding with the substr ate, suggest that the binding of Ca2+ to PLA(2) induces conformational changes at the active site and substrate-binding site. However, the s maller atomic radius with Mg2+ or the bigger atomic radii with Sr2+ an d Ba2+ might render the conformation improperly rearranged after their binding to PLA(2) molecule.