Ahmed S Ashraf, McPhie Peter, Smith Leonard A
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.
Biochemistry. 2003 Nov 4;42(43):12539-49. doi: 10.1021/bi030062c.
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.
肉毒杆菌A神经毒素的锌内肽酶轻链会发生自催化断裂，金属辅因子锌的存在会加速这种断裂[艾哈迈德，S. A.等人（2001年）《蛋白质化学杂志》20卷，221 - 231页]。我们在本文中表明，在未添加锌的情况下获得的>95%断裂轻链对源自SNAP - 25的合成肽底物保留了100%的原始催化活性。在氯化锌存在的情况下，当>95%的轻链发生自催化断裂时，制剂保留了其原始催化活性的35%。另一方面，在甘油存在的情况下，轻链未显示出自催化作用，并保留了100%的原始活性。这些结果表明，与锌一起孵育导致的活性丧失不是自催化的直接结果，并且活性位点的环境并未因断裂而受到显著影响。自催化的最佳pH值4.2 - 4.6与内在催化活性的最佳pH值（pH 7.3）不同。催化活性的竞争性抑制剂在低pH值下对自催化的抑制排除了污染蛋白酶的存在，但表明存在低pH值诱导的适合自催化的构象变化的限速步骤。我们关于断裂反应的轻链浓度依赖性的结果表明，自催化通过分子内和分子间机制发生。
