Functional involvement of Lys-6 in the enzymatic activity of phospholipase A2 from Bungarus multicinctus (Taiwan banded krait) snake venom.
Phospholipase A2 (PLA2) 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) PLA2 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-PLA2 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 PLA2. Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA2 and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA2 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 PLA2 were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA2 to 32% of that of PLA2. It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA2 activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA2 is almost the same as that of native PLA2. These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA2, and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to the distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA2.
Chang
L S
LS
Department of Biochemistry, Kaohsiung Medical College, Taiwan.
Kuo
K W
KW
Lin
S R
SR
Chang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D004546:Elapid Venoms / Q000737:chemistry*; D005453:Fluorescence; D008239:Lysine; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry* / Q000502:physiology; D054467:Phospholipases A2; D013329:Structure-Activity Relationship
D004546:Elapid Venoms; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine