De Lauzon S, Quilez R, Lion L, Desfosses B, Desfosses B, Lee I, Sari M A, Benkovic S J, Mansuy D, Mahy J P
Université René Descartes, URA 400 CNRS, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Paris, France.
Eur J Biochem. 1998 Oct 1;257(1):121-30. doi: 10.1046/j.1432-1327.1998.2570121.x.
The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)-alpha,alpha,alpha,beta-meso-tetrakis(ortho-carboxyphenyl)porph yrin [alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin]], and which exhibit in the presence of this alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] cofactor a peroxidase activity. A comparison of the dissociation constants of the complexes of 13G10 and 14H7 with various tetra-aryl-substituted porphyrin has shown that: (a) the central iron(III) atom of alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] is not recognized by either of the two antibodies; and (b) the ortho-carboxylate substituents of the meso-phenyl rings of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] are essential for the recognition of the porphyrin by 13G10 and 14H7. Measurement of the dissociation constants for the complexes of 13G10 and 14H7 with the four atropoisomers of (o-COOHPh)4-porphyrinH2 as well as mono- and di-ortho-carboxyphenyl-substituted porphyrins suggests that the three carboxylates in the alpha, alpha, beta position are recognized by both 13G10 and 14H7 with the two in the alpha, beta positions more strongly bound to the antibody protein. Accordingly, the topology of the active site of 13G10 and 14H7 has roughly two-thirds of the alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] cofactor inserted into the binding site of the antibodies, with one of the aryl ring remaining outside. Three of the carboxylates are bound to the protein but no amino acid residue acts as an axial ligand to the iron atom. Chemical modification of lysine, histidine, tryptophan and arginine residues has shown that only modification of arginine residues causes a decrease in both the binding of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] and the peroxidase activity of both antibodies. Consequently, at least one of the carboxylates of the hapten is bound to an arginine residue and no amino acids such as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O-O bond of H2O2. In addition, the amino acid sequence of both antibodies not only reveals the presence of arginine residues, which could be those involved in the binding of the carboxylates of the hapten, but also the presence of several amino acids in the complementary determining regions which could bind other carboxylates through a network of H bonds.
针对铁（III）-α，α，α，β-内消旋四（邻羧基苯基）卟啉[α，α，α，β-Fe[（o-COOHPh）4-卟啉]]产生的两种单克隆抗体13G10和14H7，其结合位点的拓扑结构已被研究，并且在该α，α，α，β-Fe[（o-COOHPh）4-卟啉]辅因子存在下表现出过氧化物酶活性。13G10和14H7与各种四芳基取代卟啉的复合物的解离常数比较表明：（a）α，α，α，β-Fe[（o-COOHPh）4-卟啉]的中心铁（III）原子未被这两种抗体中的任何一种识别；（b）α，α，α，β-Fe[（o-COOHPh）4-卟啉]的中位苯环的邻羧基取代基对于13G10和14H7识别卟啉至关重要。对13G10和14H7与（o-COOHPh）4-卟啉H2的四种阻转异构体以及单和二邻羧基苯基取代的卟啉的复合物的解离常数的测量表明，α，α，β位上的三个羧酸盐被13G10和14H7两者识别，其中α，β位上的两个与抗体蛋白结合更强。因此，13G10和14H7活性位点的拓扑结构大致有三分之二的α，α，α，β-Fe[（o-COOHPh）4-卟啉]辅因子插入到抗体的结合位点中，其中一个芳环留在外面。三个羧酸盐与蛋白质结合，但没有氨基酸残基作为铁原子的轴向配体。赖氨酸、组氨酸、色氨酸和精氨酸残基的化学修饰表明，只有精氨酸残基的修饰会导致α，α，α，β-Fe[（o-COOHPh）4-卟啉]的结合以及两种抗体的过氧化物酶活性降低。因此，半抗原的至少一个羧酸盐与精氨酸残基结合，并且没有诸如赖氨酸、组氨酸或色氨酸之类的氨基酸参与H2O2的O-O键异裂催化。此外，两种抗体的氨基酸序列不仅揭示了可能参与半抗原羧酸盐结合的精氨酸残基的存在，还揭示了互补决定区中可能通过氢键网络结合其他羧酸盐的几种氨基酸的存在。
