Protein Chemistry Department, Federal Institution "State Research Institute of Genetics and Selection of Industrial Microorganisms of the National Research Center "Kurchatov Institute", Moscow, Russia.
Protein Factory, National Research Centre "Kurchatov Institute", Moscow, Russia.
PLoS One. 2019 Dec 30;14(12):e0226636. doi: 10.1371/journal.pone.0226636. eCollection 2019.
The carboxypeptidase T (CPT) from Thermoactinomyces vulgaris has an active site structure and 3D organization similar to pancreatic carboxypeptidases A and B (CPA and CPB), but differs in broader substrate specificity. The crystal structures of CPT complexes with the transition state analogs N-sulfamoyl-L-leucine and N-sulfamoyl-L-glutamate (SLeu and SGlu) were determined and compared with previously determined structures of CPT complexes with N-sulfamoyl-L-arginine and N-sulfamoyl-L-phenylalanine (SArg and SPhe). The conformations of residues Tyr255 and Glu270, the distances between these residues and the corresponding ligand groups, and the Zn-S gap between the zinc ion and the sulfur atom in the ligand's sulfamoyl group that simulates a distance between the zinc ion and the tetrahedral sp3-hybridized carbon atom of the converted peptide bond, vary depending on the nature of the side chain in the substrate's C-terminus. The increasing affinity of CPT with the transition state analogs in the order SGlu, SArg, SPhe, SLeu correlates well with a decreasing Zn-S gap in these complexes and the increasing efficiency of CPT-catalyzed hydrolysis of the corresponding tripeptide substrates (ZAAL > ZAAF > ZAAR > ZAAE). Thus, the side chain of the ligand that interacts with the primary specificity pocket of CPT, determines the geometry of the transition complex, the relative orientation of the bond to be cleaved by the catalytic groups of the active site and the catalytic properties of the enzyme. In the case of CPB, the relative orientation of the catalytic amino acid residues, as well as the distance between Glu270 and SArg/SPhe, is much less dependent on the nature of the corresponding side chain of the substrate. The influence of the nature of the substrate side chain on the structural organization of the transition state determines catalytic activity and broad substrate specificity of the carboxypeptidase T.
耐热栖热菌羧肽酶 T（CPT）具有与胰羧肽酶 A 和 B（CPA 和 CPB）相似的活性位点结构和 3D 组织，但在更广泛的底物特异性上有所不同。已确定 CPT 与过渡态类似物 N-磺酰基-L-亮氨酸和 N-磺酰基-L-谷氨酸（SLeu 和 SGlu）的复合物的晶体结构，并与先前确定的 CPT 与 N-磺酰基-L-精氨酸和 N-磺酰基-L-苯丙氨酸（SArg 和 SPhe）的复合物结构进行了比较。残基 Tyr255 和 Glu270 的构象、这些残基与相应配体基团之间的距离以及锌离子与配体磺酰胺基团中模拟转换肽键的四面体 sp3 杂化碳原子之间距离的锌-S 间隙因底物 C 末端侧链的性质而异。CPT 与过渡态类似物的亲和力增加顺序为 SGlu、SArg、SPhe、SLeu，这与这些复合物中 Zn-S 间隙减小以及 CPT 催化水解相应三肽底物的效率增加（ZAAL>ZAAF>ZAA>ZAAE）密切相关。因此，与 CPT 的主要特异性口袋相互作用的配体侧链决定了过渡态复合物的几何形状、活性位点催化基团要切割的键的相对取向以及酶的催化特性。在 CPB 的情况下，催化氨基酸残基的相对取向以及 Glu270 和 SArg/SPhe 之间的距离对底物相应侧链的性质的依赖性要小得多。底物侧链的性质对过渡态结构组织的影响决定了羧肽酶 T 的催化活性和广泛的底物特异性。
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