Li L, Binz T, Niemann H, Singh B R
Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, Dartmouth, Massachusetts 02747, USA.
Biochemistry. 2000 Mar 7;39(9):2399-405. doi: 10.1021/bi992321x.
Type A botulinum neurotoxin (BoNT/A) is a zinc endopeptidase that contains the consensus sequence HEXXH (residues 223-227) in the toxic light chain (LC). The X-ray structure of the toxin has predicted that the two histidines of this motif are two of the three zinc-coordinating ligands and that the glutamate is a crucial amino acid involved in catalysis. The functional implication of E224 in the motif of LC was investigated by replacing the residue with glutamine and aspartate using site-directed mutagenesis. Substitution of Glu-224 with Gln (E224Q) resulted in a total loss of the endopeptidase activity, whereas substitution with Asp (E224D) retained about 1.4% of the enzymatic activity (k(cat) 140 vs 1.9 min(-1), respectively). However, K(m) values for wild-type and E224D BoNT/A LC were similar, 42 and 50 microM, respectively. Global structure, in terms of secondary structure content and topography of aromatic amino residues, Zn(2+) content, and substrate binding ability are retained in the enzymatically inactive mutants. Titration of Zn(2+) to EDTA-treated wild-type and mutant proteins indicated identical enthalpy for Zn(2+) binding. These results suggest an essential and direct role of the carboxyl group of Glu-224 in the hydrolysis of the substrate. The location of the carboxyl group at a precise position is critical for the enzymatic activity, as replacement of Glu-224 with Asp resulted in almost total loss of the activity.
A型肉毒杆菌神经毒素（BoNT/A）是一种锌内肽酶，其毒性轻链（LC）中含有共有序列HEXXH（第223 - 227位氨基酸残基）。毒素的X射线结构预测，该基序中的两个组氨酸是三个锌配位配体中的两个，而谷氨酸是参与催化的关键氨基酸。通过定点诱变将该残基替换为谷氨酰胺和天冬氨酸，研究了轻链基序中E224的功能意义。将Glu - 224替换为Gln（E224Q）导致内肽酶活性完全丧失，而替换为Asp（E224D）则保留了约1.4%的酶活性（催化常数分别为140和1.9 min⁻¹）。然而，野生型和E224D BoNT/A轻链的米氏常数（Kₘ）值相似，分别为42和50 μM。在二级结构含量、芳香族氨基酸残基拓扑结构、锌离子含量和底物结合能力方面，酶无活性的突变体保留了整体结构。用锌离子滴定经EDTA处理的野生型和突变型蛋白表明，锌离子结合的焓相同。这些结果表明，Glu - 224的羧基在底物水解中起重要且直接的作用。羧基位于精确位置对酶活性至关重要，因为用Asp替换Glu - 224几乎导致活性完全丧失。
