Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity.
Ammodytoxins (Atxs) are group II phospholipases A(2) (PLA(2)s) with presynaptic toxicity from venom of the snake Vipera ammodytes ammodytes. The molecular basis of their neurotoxicity, and that of similar PLA(2) toxins, is still to be explained. To address this problem, a surface-exposed aromatic residue, Phe(24), in the N-terminal region of the most potent Atx, AtxA, was replaced by other aromatic (tyrosine, tryptophan), hydrophobic (alanine) and polar uncharged (serine, asparagine) residues. The mutants were produced in the bacterial expression system, refolded in vitro and purified to homogeneity. All but the Trp(24) mutant, whose activity was similar to that of the wild type, showed a considerable decrease (40-80%) in enzymic activity on a micellar phosphatidylcholine substrate. This result indicates an important role for the aromatic side chains of phenylalanine or tryptophan, but not tyrosine, in PLA(2) activity, very likely at a stage of interfacial adsorption of the enzyme to zwitterionic aggregated substrates. The substitutions of Phe(24) also significantly decreased toxicity in mice, with the most prominent decrease, of 130-fold, observed in the case of the Asn(24) mutant. The results with the mutants show that there is no correlation between enzymic activity, lethality and binding affinity for three AtxA neuronal receptors (R180, R25 and calmodulin). Our results suggest a critical involvement of Phe(24) in the neurotoxicity of AtxA, apparently at a stage which does not involve the interaction with the known Atx-binding neuronal proteins and catalytic activity.
Petan
Toni
T
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
Krizaj
Igor
I
Gubensek
Franc
F
Pungercar
Joze
J
D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000187:drug effects; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D013552:Swine; D014757:Viper Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity
D002147:Calmodulin; D009498:Neurotoxins; D011994:Recombinant Proteins; D014757:Viper Venoms; C068117:ammodytoxin A; D010741:Phospholipases A