Tryptophan residues of phospholipase A2 from the venom of an Australian elapid snake (Pseudechis australis).
Tryptophan residues 31 and 69 (Trp-31 and Trp-69) in phospholipase A2 (Pa-11) from the venom of an Australian elapid snake, Pseudechis australis, were modified with N-bromosuccinimide (NBS) or with 2-nitrophenylsulphenylchloride (NPSC1). NBS oxidized only Trp-31, whereas NPSC1 reacted with both Trp-31 and Trp-69. Treatment of the enzyme with NBS at various pH values resulted in losses of enzymic and lethal activities. No protective effect on the oxidation with NBS was observed by the addition of calcium ion (20 mM) or lecithin (4 mM). The observations suggest that Trp-31 is exposed to the surface of the molecule, composes a part of the lipid-water interface recognition site around the active site and is essential for enzymic activity. Calcium ion addition to the solution caused a change in ultraviolet spectrum of the native enzyme Pa-11. The difference spectrum indicates that a charge effect caused a typical tryptophan blue shift in the Ca(2+)-enzyme complex. Pa-11 oxidized with NBS showed a smaller ultraviolet absorption difference on the addition of Ca2+ ion. The results show that the hypochromic effect induced upon the binding of Ca2+ is due to perturbation of the specific tryptophan residue (Trp-31) which is involved in the active site. Dissociation constant, Kd, of the Ca(2+)-enzyme complex was calculated to be 3.4 x 10(-4) M at pH 8.0.
Nishida
S
S
Department of Chemistry, Faculty of Science, Tohoku University, Sendai, Japan.
Tamiya
N
N
D000596:Amino Acids / Q000032:analysis; D001315:Australia; D001974:Bromosuccinimide; D002118:Calcium / Q000494:pharmacology; D004546:Elapid Venoms / Q000737:chemistry*; D007202:Indicators and Reagents; D008745:Methylation; D009578:Nitrobenzenes; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D010741:Phospholipases A / Q000737:chemistry* / Q000187:drug effects; D054467:Phospholipases A2; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000032:analysis*
D000596:Amino Acids; D004546:Elapid Venoms; D007202:Indicators and Reagents; D009578:Nitrobenzenes; D010446:Peptide Fragments; C026235:Pseudechis venom; C002732:2-nitrobenzenesulfenyl chloride; D014364:Tryptophan; D010741:Phospholipases A; D054467:Phospholipases A2; D001974:Bromosuccinimide; D002118:Calcium