Modification of tyrosine-3 (63) and lysine-6 of Taiwan cobra phospholipase A2 affects its ability to enhance 8-anilinonaphthalene-1-sulfonate fluorescence.
The Tyr-3 (63) and Lys-6 of Naja naja atra phospholipase A2 (PLA2) were modified with p-nitrobenzenesulfonyl fluoride and 4-chloro-3,5-dinitrobenzoate, respectively. Although the ability of the Lys-modified derivative to enhance the ANS fluorescence was lower than that observed with native PLA2, the ANS-binding affinity of the Lys-modified derivative was similar to that of the native enzyme. Modifications on Tyr-3 or/and Tyr-63 of PLA2 resulted in the complete loss of its ability to enhance the ANS fluorescence. Nevertheless, the extent of O-sulfonylation of Tyr residues was not effectively reduced by the addition of ANS. This suggests that Tyr-3 and Tyr-63 interact with the bound ANS in an aromatic ring-stacking manner. Alternatively, the incorporation of a carboxydinitrophenylated group on Lys-6 may notably perturb the nonpolarity of the ANS-binding site.
Department of Biochemistry, Kaohsiung Medical College, Taiwan, ROC.
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D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites; D004137:Dinitrochlorobenzene / Q000494:pharmacology; D004546:Elapid Venoms; D004795:Enzyme Stability; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008239:Lysine; D008961:Models, Structural; D009578:Nitrobenzenes / Q000494:pharmacology; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism*; D054467:Phospholipases A2; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014443:Tyrosine
D000817:Anilino Naphthalenesulfonates; D004137:Dinitrochlorobenzene; D004546:Elapid Venoms; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009578:Nitrobenzenes; C034442:4-nitrobenzenesulfonyl fluoride; D014443:Tyrosine; C027132:1-anilino-8-naphthalenesulfonate; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine