The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2.
In order to address the mechanism whereby Ca2+ wad crucial for the manifestation of the enzymatic activity of phospholipase A2 (PLA2), four divalent cations were used to assess their influences on the catalytic activity and the fine structures of Naja naja atra PLA2. It was found that substitution of Mg2+ or Sr2+ for Ca2+ in the substrate solution caused a decrease in the PLA2 activity to 77.5% or 54.5%, respectively, of that in the presence of Ca2+. However, no PLA2 activity was observed with the addition of Ba2+. With the exception of Mg2+, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA2 markedly increased with the binding of cations to PLA2. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate and p-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca2+, Sr2+, and Ba2+, but not by Mg2+. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba2+ > 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 PLA2 and that Tyr-3, Lys-6, and Tyr-63 of PLA2 are involved in the binding with the substrate, suggest that the binding of Ca2+ to PLA2 induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg2+ or the bigger atomic radii with Sr2+ and Ba2+ might render the conformation improperly rearranged after their binding to PLA2 molecule.
Chang
L S
LS
Department of Biochemistry, Kaohsiung Medical College, Taiwan, ROC.
Lin
S R
SR
Chang
C C
CC
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001464:Barium / Q000494:pharmacology; D001557:Benzenesulfonates; D002118:Calcium / Q000494:pharmacology*; D004546:Elapid Venoms / Q000201:enzymology*; D005456:Fluorescent Dyes; D008239:Lysine / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013324:Strontium / Q000494:pharmacology; D014302:Trinitrobenzenesulfonic Acid / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D001557:Benzenesulfonates; D004546:Elapid Venoms; D005456:Fluorescent Dyes; C081297:4-nitrobenzenesulfonate; D001464:Barium; D014443:Tyrosine; C027132:1-anilino-8-naphthalenesulfonate; D014302:Trinitrobenzenesulfonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D008274:Magnesium; D008239:Lysine; D002118:Calcium; D013324:Strontium