Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Aug 1;373(Pt 3):933–939. doi: 10.1042/BJ20030342

Subsite specificity (S3, S2, S1', S2' and S3') of oligopeptidase B from Trypanosoma cruzi and Trypanosoma brucei using fluorescent quenched peptides: comparative study and identification of specific carboxypeptidase activity.

Jefferson P Hemerly 1, Vitor Oliveira 1, Elaine Del Nery 1, Rory E Morty 1, Norma W Andrews 1, Maria A Juliano 1, Luiz Juliano 1
PMCID: PMC1223545  PMID: 12737623

Abstract

We characterized the extended substrate binding site of recombinant oligopeptidase B enzymes from Trypanosoma cruzi (Tc-OP) and Trypanosoma brucei (Tb-OP), evaluating the specificity of their S3, S2, S1', S2' and S3' subsites. Five series of internally quenched fluorescent peptides based on the substrate Abz-AGGRGAQ-EDDnp [where Abz is o -aminobenzoic acid and EDDnp is N -(2,4-dinitrophenyl)ethylenediamine] were designed to contain amino acid residues with side chains of a minimum size, and each residue position of this substrate was modified. Synthetic peptides of different lengths derived from the human kininogen sequence were also examined, and peptides of up to 17 amino acids were found to be hydrolysed by Tc-OP and Tb-OP. These two oligopeptidases were essentially arginyl hydrolases, since for all peptides examined the only cleavage site was the Arg-Xaa bond. We also demonstrated that Tc-OP and Tb-OP have a very specific carboxypeptidase activity for basic amino acids, which depends on the presence of at least of a pair of basic amino acids at the C-terminal end of the substrate. The peptide with triple Arg residues (Abz-AGRRRAQ-EDDnp) was an efficient substrate for Tc-OP and Tb-OP: the Arg-Ala peptide bond was cleaved first and then two C-terminal Arg residues were successively removed. The S1' subsite seems to be an important determinant of the specificity of both enzymes, showing a preference for Tyr, Ser, Thr and Gln as hydrogen donors. The presence of these amino acids at P1' resulted in substrates that were hydrolysed with K (m) values in the sub-micromolar range. Taken together, this work supports the view that oligopeptidase B is a specialized protein-processing enzyme with a specific carboxypeptidase activity. Excellent substrates were obtained for Tb-OP and Tc-OP (Abz-AMRRTISQ-EDDnp and Abz-AHKRYSHQ-EDDnp respectively), which were hydrolysed with remarkably high k (cat) and low K (m) values.

Full Text

The Full Text of this article is available as a PDF (119.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Araujo M. C., Melo R. L., Cesari M. H., Juliano M. A., Juliano L., Carmona A. K. Peptidase specificity characterization of C- and N-terminal catalytic sites of angiotensin I-converting enzyme. Biochemistry. 2000 Jul 25;39(29):8519–8525. doi: 10.1021/bi9928905. [DOI] [PubMed] [Google Scholar]
  2. Burleigh B. A., Caler E. V., Webster P., Andrews N. W. A cytosolic serine endopeptidase from Trypanosoma cruzi is required for the generation of Ca2+ signaling in mammalian cells. J Cell Biol. 1997 Feb 10;136(3):609–620. doi: 10.1083/jcb.136.3.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Caler E. V., Vaena de Avalos S., Haynes P. A., Andrews N. W., Burleigh B. A. Oligopeptidase B-dependent signaling mediates host cell invasion by Trypanosoma cruzi. EMBO J. 1998 Sep 1;17(17):4975–4986. doi: 10.1093/emboj/17.17.4975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fenno J. C., Lee S. Y., Bayer C. H., Ning Y. The opdB locus encodes the trypsin-like peptidase activity of Treponema denticola. Infect Immun. 2001 Oct;69(10):6193–6200. doi: 10.1128/IAI.69.10.6193-6200.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fülöp V., Böcskei Z., Polgár L. Prolyl oligopeptidase: an unusual beta-propeller domain regulates proteolysis. Cell. 1998 Jul 24;94(2):161–170. doi: 10.1016/s0092-8674(00)81416-6. [DOI] [PubMed] [Google Scholar]
  6. Gérczei T., Keserü G. M., Náray-Szabó G. Construction of a 3D model of oligopeptidase B, a potential processing enzyme in prokaryotes. J Mol Graph Model. 2000 Feb;18(1):7-17, 57-8. doi: 10.1016/s1093-3263(99)00042-x. [DOI] [PubMed] [Google Scholar]
  7. Hasebe T., Hua J., Someya A., Morain P., Checler F., Nagaoka I. Involvement of cytosolic prolyl endopeptidase in degradation of p40-phox splice variant protein in myeloid cells. J Leukoc Biol. 2001 Jun;69(6):963–968. [PubMed] [Google Scholar]
  8. Heiman C., Miller C. G. Salmonella typhimurium mutants lacking protease II. J Bacteriol. 1978 Aug;135(2):588–594. doi: 10.1128/jb.135.2.588-594.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jameson G. W., Roberts D. V., Adams R. W., Kyle W. S., Elmore D. T. Determination of the operational molarity of solutions of bovine alpha-chymotrypsin, trypsin, thrombin and factor Xa by spectrofluorimetric titration. Biochem J. 1973 Jan;131(1):107–117. doi: 10.1042/bj1310107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kanatani A., Masuda T., Shimoda T., Misoka F., Lin X. S., Yoshimoto T., Tsuru D. Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme. J Biochem. 1991 Sep;110(3):315–320. doi: 10.1093/oxfordjournals.jbchem.a123577. [DOI] [PubMed] [Google Scholar]
  11. Leite M. F., Moyer M. S., Andrews N. W. Expression of the mammalian calcium signaling response to Trypanosoma cruzi in Xenopus laevis oocytes. Mol Biochem Parasitol. 1998 Apr 1;92(1):1–13. doi: 10.1016/s0166-6851(97)00211-9. [DOI] [PubMed] [Google Scholar]
  12. Morty R. E., Lonsdale-Eccles J. D., Mentele R., Auerswald E. A., Coetzer T. H. Trypanosome-derived oligopeptidase B is released into the plasma of infected rodents, where it persists and retains full catalytic activity. Infect Immun. 2001 Apr;69(4):2757–2761. doi: 10.1128/IAI.69.4.2757-2761.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Morty R. E., Lonsdale-Eccles J. D., Morehead J., Caler E. V., Mentele R., Auerswald E. A., Coetzer T. H., Andrews N. W., Burleigh B. A. Oligopeptidase B from Trypanosoma brucei, a new member of an emerging subgroup of serine oligopeptidases. J Biol Chem. 1999 Sep 10;274(37):26149–26156. doi: 10.1074/jbc.274.37.26149. [DOI] [PubMed] [Google Scholar]
  14. Morty R. E., Troeberg L., Pike R. N., Jones R., Nickel P., Lonsdale-Eccles J. D., Coetzer T. H. A trypanosome oligopeptidase as a target for the trypanocidal agents pentamidine, diminazene and suramin. FEBS Lett. 1998 Aug 21;433(3):251–256. doi: 10.1016/s0014-5793(98)00914-4. [DOI] [PubMed] [Google Scholar]
  15. Morty R. E., Troeberg L., Powers J. C., Ono S., Lonsdale-Eccles J. D., Coetzer T. H. Characterisation of the antitrypanosomal activity of peptidyl alpha-aminoalkyl phosphonate diphenyl esters. Biochem Pharmacol. 2000 Nov 15;60(10):1497–1504. doi: 10.1016/s0006-2952(00)00459-7. [DOI] [PubMed] [Google Scholar]
  16. Morty Rory E., Fülöp Vilmos, Andrews Norma W. Substrate recognition properties of oligopeptidase B from Salmonella enterica serovar Typhimurium. J Bacteriol. 2002 Jun;184(12):3329–3337. doi: 10.1128/JB.184.12.3329-3337.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Oleksyszyn J., Boduszek B., Kam C. M., Powers J. C. Novel amidine-containing peptidyl phosphonates as irreversible inhibitors for blood coagulation and related serine proteases. J Med Chem. 1994 Jan 21;37(2):226–231. doi: 10.1021/jm00028a004. [DOI] [PubMed] [Google Scholar]
  18. Pacaud M., Richaud C. Protease II from Escherichia coli. Purification and characterization. J Biol Chem. 1975 Oct 10;250(19):7771–7779. [PubMed] [Google Scholar]
  19. Polgár L., Felföldi F. Oligopeptidase B: cloning and probing stability under nonequilibrium conditions. Proteins. 1998 Mar 1;30(4):424–434. [PubMed] [Google Scholar]
  20. Polgár L. Oligopeptidase B: a new type of serine peptidase with a unique substrate-dependent temperature sensitivity. Biochemistry. 1999 Nov 23;38(47):15548–15555. doi: 10.1021/bi991767a. [DOI] [PubMed] [Google Scholar]
  21. Rawlings N. D., Barrett A. J. MEROPS: the peptidase database. Nucleic Acids Res. 2000 Jan 1;28(1):323–325. doi: 10.1093/nar/28.1.323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  23. Troeberg L., Pike R. N., Morty R. E., Berry R. K., Coetzer T. H., Lonsdale-Eccles J. D. Proteases from Trypanosoma brucei brucei. Purification, characterisation and interactions with host regulatory molecules. Eur J Biochem. 1996 Jun 15;238(3):728–736. doi: 10.1111/j.1432-1033.1996.0728w.x. [DOI] [PubMed] [Google Scholar]
  24. WILKINSON G. N. Statistical estimations in enzyme kinetics. Biochem J. 1961 Aug;80:324–332. doi: 10.1042/bj0800324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yoshimoto T., Tabira J., Kabashima T., Inoue S., Ito K. Protease II from Moraxella lacunata: cloning, sequencing, and expression of the enzyme gene, and crystallization of the expressed enzyme. J Biochem. 1995 Mar;117(3):654–660. doi: 10.1093/oxfordjournals.jbchem.a124759. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES