IDENTIFICATION OF FUNCTIONAL INVOLVEMENT OF TRYPTOPHAN RESIDUES IN PHOSPHOLIPASE-A(2) FROM NAJA-NAJA-ATRA (TAIWAN COBRA) SNAKE-VENOM

Phospholipase A2 (PLA2) from Naja naja atra snake venom was subjected to Trp modification with 2-nitrophenylsulfenyl chloride (NPS-Cl), and six derivatives were separated by HPLC. The results of amino-acid analysis and sequence determination revealed that Trp-18, Trp-19 and Trp-61 were modified by NPS-Cl. The order of accessibilities of the three Trp residues for NPS-Cl was Trp-18 > Trp-19 > Trp-61. Sulfenylation of Trp-18 caused a 92% drop in enzymatic activity. Modification of Trp-19 and Trp-61 resulted in a decrease in enzymatic activity of PLA2 by 45.5% and 51%, respectively. The enzyme modified on both Trp-18 and Trp-19 or on both Trp-18 and Trp-61 retained little PLA2 activity. It is evident that Trp-18 plays a more crucial function in PLA2 than Trp-19 and Trp-61. Sulfenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca2+ binding and antigenicity of sulfenylated PLA2 was unaffected. These observations, together with the fact that Trp-18 is involved in the substrate binding of PLA2, suggest that incorporation of a bulky NPS group on Trp-18 might give rise to a direct distortion of the interaction between substrate and the enzyme molecule. Alternatively, modification of Trp-19 and Trp-61 might indirectly affect the interfacial binding of PLA2 with its substrate.