Autocatalytically fragmented light chain of botulinum a neurotoxin is enzymatically active.
The zinc-endopeptidase light chain of botulinum A neurotoxin undergoes autocatalytic fragmentation that is accelerated by the presence of the metal cofactor, zinc [Ahmed, S. A. et al. (2001) J. Protein Chem. 20, 221-231]. We show in this paper that >95% fragmented light chain obtained in the absence of added zinc retained 100% of its original catalytic activity against a SNAP-25-derived synthetic peptide substrate. In the presence of zinc chloride, when >95% of the light chain had undergone autocatalytic fragmentation, the preparation retained 35% of its original catalytic activity. On the other hand, in the presence of glycerol, the light chain did not display autocatalysis and retained 100% of the original activity. These results suggest that the activity loss by incubation with zinc was not a direct consequence of autocatalysis and that the environment of the active site was not affected significantly by the fragmentation. The optimum pH 4.2-4.6 for autocatalysis was different than that (pH 7.3) for intrinsic catalytic activity. Inhibition of autocatalysis at low pH by a competitive inhibitor of catalytic activity rules out the presence of a contaminating protease but suggests a rate-limiting step of low pH-induced conformational change suitable for autocatalysis. Our results of LC concentration dependence of the fragmentation reaction indicate that the autocatalysis occurs by both intramolecular and intermolecular mechanisms.
Ahmed
S Ashraf
SA
Department of Immunology and Molecular Biology, Division of Toxinology and Aerobiology, U. S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702, USA. syed.ahmed@amedd.army.mil
McPhie
Peter
P
Smith
Leonard A
LA
D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data
D001905:Botulinum Toxins