Tryptophan 110, a residue involved in the toxic activity but not in the enzymatic activity of notexin.
We prepared two derivatives of notexin, a phospholipase A2 from Notechis scutatus scutatus venom, by modifying the protein with 2-nitrophenylsulfenylchloride, a tryptophan-specific reagent. One derivative was modified at both tryptophans 20 and 110 whereas the other was modified at tryptophan 20. Evidence based on circular dichroic analysis and antigenicity towards a notexin-specific monoclonal antibody indicated that derivatization at both tryptophans did not affect the tertiary structure of notexin. Concomitant modification of tryptophans 20 and 110 induced a marked decrease in the capacity of notexin to kill mice and to block neuromuscular transmission in the chick biventer cervicis preparation, whereas selective modification at tryptophan 20 had no effect on the lethal properties of notexin. This implies that the decrease in the lethal properties of notexin after derivatization was due to modification at tryptophan 110. However, the diderivatized notexin retained full enzymatic activity, implying that neither tryptophan 20 and tryptophan 110 are involved in the catalytic function of the molecule. We conclude that notexin harbours two functional sites. One of them corresponds to the enzymatic site, whereas the other, which includes tryptophan 110, provides specific toxic characteristics to notexin. By reference to previous crystallographic studies, the relative spatial positions of elements involved in toxicity and the catalytic site, we propose a possible orientation of notexin with respect to its putative membrane-bound target.
Mollier
P
P
Département de Biologie, Centre d'Etudes Nucléaires de Saclay, Gif-sur-Yvette, France.
Chwetzoff
S
S
Bouet
F
F
Harvey
A L
AL
Ménez
A
A
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004546:Elapid Venoms / Q000378:metabolism* / Q000633:toxicity; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000378:metabolism* / Q000633:toxicity; D009578:Nitrobenzenes; D011863:Radioimmunoassay; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000378:metabolism*
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D004546:Elapid Venoms; D009498:Neurotoxins; D009578:Nitrobenzenes; C005082:notexin; C002732:2-nitrobenzenesulfenyl chloride; D014364:Tryptophan