FUNCTIONAL INVOLVEMENT OF LYS-6 IN THE ENZYMATIC-ACTIVITY OF PHOSPHOLIPASE-A(2) FROM BUNGARUS-MULTICINCTUS (TAIWAN BANDED KRAIT) SNAKE-VENOM

Phospholipase A(2) (PLA(2)) from Bungarus multicinctus snake venom was subjected to Lys modification with 4-chloro-3,5-dinitrobenzoate and trinitrobenzene sulfonic acid, and one major carboxydinitrophenylated (CDNP) PLA(2) and two trinitrophenylated (TNP) derivatives (TNP-1 and TNP-2) were separated by high-performance liquid chromatography. The results of amino acid analysis and sequence determination revealed that CDNP-PLA(2) and TNP-1 contained one modified Lys residue at position 6, and both Lys-6 and Lys-62 were modified in TNP-2. It seemed that the Lys-6 was more accessible to modified reagents than other Lys residues in PLA(2). Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA(2) and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA(2) activity. Either carboxydinitrophenylation or trinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca2+ binding and antigenicity of Lys-6-modified PLA(2) were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA(2) to 32% of that of PLP(2). It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA(2) activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA(2) is almost the same as that of native PLA(2). These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA(2), and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to a distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA(2).