The nature of the ligand’s side chain interacting with the S1'-subsite of metallocarboxypeptidase T (from Thermoactinomyces vulgaris) determines the geometry of the tetrahedral transition complex

Autoři: Valery Kh. Akparov aff001;  Vladimir I. Timofeev aff003;  Galina E. Konstantinova aff001;  Ilyas G. Khaliullin aff005;  Inna P. Kuranova aff003;  Tatiana V. Rakitina aff002;  Vytas Švedas aff007
Působiště autorů: Protein Chemistry Department, Federal Institution "State Research Institute of Genetics and Selection of Industrial Microorganisms of the National Research Center "Kurchatov Institute", Moscow, Russia aff001;  Protein Factory, National Research Centre “Kurchatov Institute”, Moscow, Russia aff002;  Laboratory of X-ray analysis methods and synchrotron radiation, Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences, Moscow, Russia aff003;  Kurchatov center of synchrotron-neutron research, National Research Centre “Kurchatov Institute”, Moscow, Russia aff004;  Laboratory of ion and molecular physics, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow region, Russia aff005;  Laboratory of Hormonal Regulation Proteins, Shemyakin−Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia aff006;  Faculty of Bioengineering and Bioinformatics, Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Moscow, Russia aff007
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
prolekare.web.journal.doi_sk: 10.1371/journal.pone.0226636


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.

Klíčová slova:

Crystal structure – Enzyme structure – Enzymes – Ethers – Mixtures – Thin-layer chromatography – Transition state – Zinc


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