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

Souhrn

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


Zdroje

1. Turk B. Targeting proteases: Successes, failures and future prospects. Nat Rev Drug Discov. 2006;5(9):785–99. doi: 10.1038/nrd2092 16955069

2. Sapio MR, Fricker LD. Carboxypeptidases in disease: Insights from peptidomic studies. Proteomics—Clin Appl. 2014;8(5–6):327–37. doi: 10.1002/prca.201300090 24470285

3. Lyons PJ, Callaway MB, Fricker LD. Characterization of carboxypeptidase A6, an extracellular matrix peptidase. J Biol Chem. 2008;283:7054–7063. doi: 10.1074/jbc.M707680200 18178555

4. Szeto MWY, Mujika JI, Zurek J, Mulholland AJ, Harvey JN. QM/MM study on the mechanism of peptide hydrolysis by carboxypeptidase A. J Mol Struct THEOCHEM. 2009;898:106–114.

5. Wu S, Zhang C, Xu D, Guo H. Catalysis of Carboxypeptidase A: Promoted-Water versus Nucleophilic Pathways. J Phys Chem B. 2010;114:9259–9267. doi: 10.1021/jp101448j 20583802

6. Fernandez D, Boix E, Pallares I, Aviles FX, Vendrell J. Analysis of a new crystal form of procarboxypeptidase B: further insights into the catalytic mechanism. Biopolymers. 2009;93(2):178–85.

7. Cho JH, Kim DH, Lee KJ, Choi KY. The role of Tyr248 probed by mutant bovine carboxypeptidase A: insight into the catalytic mechanism of carboxypeptidase A. Biochemistry. 2001;40(34):10197–203. doi: 10.1021/bi010807j 11513597

8. Gardell S, Craik C, Hilvert D, Urdea M, Rutter WJ. Site-directed mutagenesis shows that tyrosine 248 of carboxypeptidase A does not play a crucial role in catalysis. Nature. 1985;317(6037):551–5. doi: 10.1038/317551a0 3840231

9. Lipscomb WM, Strater N. Recent Advances in Zinc Enzymology. Chem Rev. 1996;96:2375–2433. doi: 10.1021/cr950042j 11848831

10. Christianson DW, Lipscomb WN. Carboxypeptidase A. Acc ChemRes. 1989;22:62–9.

11. Akparov V, Timofeev V, Khaliullin I, Švedas V, Kuranova I, Rakitina T. Crystal structures of carboxypeptidase T complexes with transition-state analogs. J Biomol Struct Dyn. 2018;36(15):3958–66. doi: 10.1080/07391102.2017.1404932 29129130

12. Gutteridge A, Thornton J. Conformational change in substrate binding, catalysis and product release: an open and shut case? FEBS Lett. 2004;567:67–73. doi: 10.1016/j.febslet.2004.03.067 15165895

13. Gutteridge A, Thornton J. Conformational changes observed in enzyme crystal structures upon substrate binding. J Mol Biol; 2005;346(1):21–28. doi: 10.1016/j.jmb.2004.11.013 15663924

14. Brylinski M, Skolnick J. What is the relationship between the global structures of apo and holo proteins? Proteins. 2008;70(2):363–377. doi: 10.1002/prot.21510 17680687

15. Clark JJ, Benson ML, Smith RD, Carlson HA. Inherent versus induced protein flexibility: Comparisons within and between apo and holo structures. PLoS Comput Biol. 2019;15(1):e1006705. doi: 10.1371/journal.pcbi.1006705 30699115

16. Akparov VK, Timofeev VI, Khaliullin IG, Švedas V, Chestukhina GG, Kuranova IP. Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris. FEBS J [Internet]. 2015;282(7):1214–24. Available from: doi: 10.1111/febs.13210 25619204

17. Stepanov VM. Carboxypeptidase T. Methods Enzymol [Internet]. 1995 Jan [cited 2014 Nov 30];248:675–83. Available from: http://www.ncbi.nlm.nih.gov/pubmed/7674954 doi: 10.1016/0076-6879(95)48044-7 7674954

18. Akparov VK, Grishin AM, Yusupova MP, Ivanova NM, Chestukhina GG. Structural principles of the wide substrate specificity of Thermoactinomyces vulgaris carboxypeptidase T. Reconstruction of the carboxypeptidase B primary specificity pocket. Biochemistry (Mosc). 2007;72(4):416–23.

19. Schechter I, Berger A. On the size of the active site in proteases. I. Papain. 1967. Biochem Biophys Res Commun. 2012;425(3):497–502. doi: 10.1016/j.bbrc.2012.08.015 22925665

20. Akparov V, Timofeev V, Kuranova I, Rakitina T. Crystal structure of mutant carboxypeptidase T from Thermoactinomyces vulgaris with an implanted S1’ subsite from pancreatic carboxypeptidase B. Acta Crystallogr F Struct Biol Commun. 2018;74(10):638–43.

21. Akparov VK, Belianova LP, Baratova LA, Stepanov VM. Subtilisin 72: a serine protease from Bac. subtilis strain 72—an enzyme similar to subtilisin Carlsberg. Biokhimiia. 1979;44(5):886–91. 110360

22. Liublinskaia L.A.Iakushcheva LD, Stepanov VM. The synthesis of the peptide substrates of the subtilisin and its analogs. Bioorg Khim. 1977;3:273–279.

23. Yusupova M, Kotlova E, Timokhina E, Stepanov V. The enzymatic synthesis of arginine peptides–chromophoric substrates of metalloproteases and carboxypeptidases. Bioorganic Chem. 1995;21:33–8.

24. Voiushina TL, Liublinskaia LA, Timokhina EA, Stepanov VM. 4. Synthesis of p-nitroanilides of acylated peptides catalyzed by thermolysin. Bioorg Khim, (Russian). 1987;13:615–622.

25. Cueni LB, Bazzone TJ, Riordan JF, Vallee BL. Affinity chromatographic sorting of carboxypeptidase A and its chemically modified derivatives. Anal Biochem. 1980;107(2):341–9. doi: 10.1016/0003-2697(80)90394-2 7435967

26. Novagen pET System Manual TB055 7th Ed. Novagen pET System Manual TB055, 7th ed. Novagen Madison WI. 1997;

27. Trachuk L, Letarov A, Kudelina IA, Yusupova MP, Chestukhina GG. In vitro refolding of carboxypeptidase T precursor from Thermoactinomyces vulgaris obtained in Escherichia coli as cytoplasmic inclusion bodies. Protein Expr Purif. 2005;40(1):51–9. doi: 10.1016/j.pep.2004.10.020 15721771

28. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. doi: 10.1006/abio.1976.9999 942051

29. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680–5. doi: 10.1038/227680a0 5432063

30. Takahashi S, Tsurumura T, Aritake K, Furubayashi N, Sato M, Yamanaka M, et al. High-quality crystals of human haematopoietic prostaglandin D synthase with novel inhibitors. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010;66(7):846–50.

31. Kuranova IP, Smirnova EA, Abramchik YA, Chupova LA, Esipov RS, Akparov VK, et al. Crystal Growth of Phosphopantetheine Adenylyltransferase, Carboxypeptidase T, and Thymidine Phosphorylase| on the International Space Station by the Capillary Counter-Diffusion Method. Crystallogr Reports. 2011;56:5884–5891.

32. McPherson A. Macromolecular crystal growth in microgravity. Crystallogr Rev. 1996;6:157–308.

33. Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997;276:307–26.

34. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr. 2007;40(4):658–74.

35. Akparov VK, Timofeev VI, Kuranova IP. Three-dimensional structure of recombinant carboxypeptidase T from Thermoactinomyces vulgaris without calcium ions. Crystallogr Reports [Internet]. 2011 Jul 28 [cited 2014 Nov 30];56(4):596–602. Available from: http://link.springer.com/10.1134/S106377451104002X

36. Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr Sect D Biol Crystallogr. 1997;53(3):240–55.

37. Emsley P, Cowtan K. Coot: Model-building tools for molecular graphics. Acta Crystallogr Sect D Biol Crystallogr. 2004;60(12 I):2126–32.

38. Cornish-Bowden A. Fundamentals of Enzyme Kinetics. 4th editio. Weinheim: Wiley-VCH; 2013.

39. Park JD, Kim DH, Kim SJ, Woo JR, Ryu SE. Sulfamide-based inhibitors for carboxypeptidase A. Novel type transition state analogue inhibitors for zinc proteases. J Med Chem. 2002;45(24):5295–302. doi: 10.1021/jm020258v 12431056

40. Akparov VK, Sokolenko N, Timofeev V, Kuranova I. Structure of the complex of carboxypeptidase B and N -sulfamoyl- L -arginine. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2015;71:1335–40.

41. Akparov V, Timofeev V, Khaliullin I, Švedas V, Kuranova I. Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine—a transition state analog of non-specific substrate. J Biomol Struct Dyn. 2018;36(4):956–65. doi: 10.1080/07391102.2017.1304242 28274181


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