Identification of functional involvement of tryptophan residues in phospholipase A2 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.
Chang
L S
LS
Department of Biochemistry, Kaohsiung Medical College, Taiwan.
Kuo
K W
KW
Chang
C C
CC
D000595:Amino Acid Sequence; D001665:Binding Sites; D004546:Elapid Venoms / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009578:Nitrobenzenes; D010446:Peptide Fragments / Q000737:chemistry; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D014357:Trypsin; D014364:Tryptophan / Q000737:chemistry*
D004546:Elapid Venoms; D009578:Nitrobenzenes; D010446:Peptide Fragments; C002732:2-nitrobenzenesulfenyl chloride; D014364:Tryptophan; D010741:Phospholipases A; D054467:Phospholipases A2; D014357:Trypsin