Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1997 Nov 17;16(22):6626–6635. doi: 10.1093/emboj/16.22.6626

Converting blood coagulation factor IXa into factor Xa: dramatic increase in amidolytic activity identifies important active site determinants.

K P Hopfner 1, H Brandstetter 1, A Karcher 1, E Kopetzki 1, R Huber 1, R A Engh 1, W Bode 1
PMCID: PMC1170267  PMID: 9362477

Abstract

The coagulation factors IXa (fIXa) and Xa (fXa) share extensive structural and functional homology; both cleave natural substrates effectively only with a cofactor at a phospholipid surface. However, the amidolytic activity of fIXa is 10(4)-fold lower than that of fXa. To identify determinants of this poor reactivity, we expressed variants of truncated fIXa (rf9a) and fXa (rf10a) in Escherichia coli. The crystal structures of fIXa and fXa revealed four characteristic active site components which were subsequently exchanged between rf9a and rf10a. Exchanging Glu219 by Gly or exchanging the 148 loop did not increase activity of rf9a, whereas corresponding mutations abolished reactivity of rf10a. Exchanging Ile213 by Val only moderately increased reactivity of rf9a. Exchanging the 99 loop, however, dramatically increased reactivity. Furthermore, combining all four mutations essentially introduced fXa properties into rf9a: the amidolytic activity was increased 130-fold with fXa substrate selectivity. The results suggest a 2-fold origin of fIXa's poor reactivity. A narrowed S3/S4 subsite disfavours interaction with substrate P3/P4 residues, while a distorted S1 subsite disfavours effective cleavage of the scissile bond. Both defects could be repaired by introducing fXa residues. Such engineered coagulation enzymes will be useful in diagnostics and in the development of therapeutics.

Full Text

The Full Text of this article is available as a PDF (514.6 KB).

Selected References

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

  1. Bajaj S. P., Rapaport S. I., Prodanos C. A simplified procedure for purification of human prothrombin, factor IX and factor X. Prep Biochem. 1981;11(4):397–412. doi: 10.1080/00327488108065531. [DOI] [PubMed] [Google Scholar]
  2. Bode W., Mayr I., Baumann U., Huber R., Stone S. R., Hofsteenge J. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 1989 Nov;8(11):3467–3475. doi: 10.1002/j.1460-2075.1989.tb08511.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brandstetter H., Bauer M., Huber R., Lollar P., Bode W. X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9796–9800. doi: 10.1073/pnas.92.21.9796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brandstetter H., Kühne A., Bode W., Huber R., von der Saal W., Wirthensohn K., Engh R. A. X-ray structure of active site-inhibited clotting factor Xa. Implications for drug design and substrate recognition. J Biol Chem. 1996 Nov 22;271(47):29988–29992. doi: 10.1074/jbc.271.47.29988. [DOI] [PubMed] [Google Scholar]
  5. Brinkmann U., Mattes R. E., Buckel P. High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product. Gene. 1989 Dec 21;85(1):109–114. doi: 10.1016/0378-1119(89)90470-8. [DOI] [PubMed] [Google Scholar]
  6. Carter P. J., Winter G., Wilkinson A. J., Fersht A. R. The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus). Cell. 1984 Oct;38(3):835–840. doi: 10.1016/0092-8674(84)90278-2. [DOI] [PubMed] [Google Scholar]
  7. Castillo M. J., Kurachi K., Nishino N., Ohkubo I., Powers J. C. Reactivity of bovine blood coagulation factor IXa beta, factor Xa beta, and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X. Biochemistry. 1983 Mar 1;22(5):1021–1029. doi: 10.1021/bi00274a004. [DOI] [PubMed] [Google Scholar]
  8. Cho K., Tanaka T., Cook R. R., Kisiel W., Fujikawa K., Kurachi K., Powers J. C. Active-site mapping of bovine and human blood coagulation serine proteases using synthetic peptide 4-nitroanilide and thio ester substrates. Biochemistry. 1984 Feb 14;23(4):644–650. doi: 10.1021/bi00299a009. [DOI] [PubMed] [Google Scholar]
  9. Dang Q. D., Guinto E. R., di Cera E. Rational engineering of activity and specificity in a serine protease. Nat Biotechnol. 1997 Feb;15(2):146–149. doi: 10.1038/nbt0297-146. [DOI] [PubMed] [Google Scholar]
  10. Davie E. W., Fujikawa K., Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry. 1991 Oct 29;30(43):10363–10370. doi: 10.1021/bi00107a001. [DOI] [PubMed] [Google Scholar]
  11. Di Scipio R. G., Hermodson M. A., Yates S. G., Davie E. W. A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S. Biochemistry. 1977 Feb 22;16(4):698–706. doi: 10.1021/bi00623a022. [DOI] [PubMed] [Google Scholar]
  12. Di Scipio R. G., Kurachi K., Davie E. W. Activation of human factor IX (Christmas factor). J Clin Invest. 1978 Jun;61(6):1528–1538. doi: 10.1172/JCI109073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Duffy E. J., Lollar P. Intrinsic pathway activation of factor X and its activation peptide-deficient derivative, factor Xdes-143-191. J Biol Chem. 1992 Apr 15;267(11):7821–7827. [PubMed] [Google Scholar]
  14. Engh R. A., Brandstetter H., Sucher G., Eichinger A., Baumann U., Bode W., Huber R., Poll T., Rudolph R., von der Saal W. Enzyme flexibility, solvent and 'weak' interactions characterize thrombin-ligand interactions: implications for drug design. Structure. 1996 Nov 15;4(11):1353–1362. doi: 10.1016/s0969-2126(96)00142-6. [DOI] [PubMed] [Google Scholar]
  15. Fujikawa K., Legaz M. E., Kato H., Davie E. W. The mechanism of activation of bovine factor IX (Christmas factor) by bovine factor XIa (activated plasma thromboplastin antecedent). Biochemistry. 1974 Oct 22;13(22):4508–4516. doi: 10.1021/bi00719a006. [DOI] [PubMed] [Google Scholar]
  16. Furie B. C., Furie B. Coagulant protein of Russell's viper venom. Methods Enzymol. 1976;45:191–205. doi: 10.1016/s0076-6879(76)45019-x. [DOI] [PubMed] [Google Scholar]
  17. Furie B., Furie B. C. The molecular basis of blood coagulation. Cell. 1988 May 20;53(4):505–518. doi: 10.1016/0092-8674(88)90567-3. [DOI] [PubMed] [Google Scholar]
  18. Gibbs C. S., Coutré S. E., Tsiang M., Li W. X., Jain A. K., Dunn K. E., Law V. S., Mao C. T., Matsumura S. Y., Mejza S. J. Conversion of thrombin into an anticoagulant by protein engineering. Nature. 1995 Nov 23;378(6555):413–416. doi: 10.1038/378413a0. [DOI] [PubMed] [Google Scholar]
  19. Grodberg J., Dunn J. J. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol. 1988 Mar;170(3):1245–1253. doi: 10.1128/jb.170.3.1245-1253.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hedstrom L., Perona J. J., Rutter W. J. Converting trypsin to chymotrypsin: residue 172 is a substrate specificity determinant. Biochemistry. 1994 Jul 26;33(29):8757–8763. doi: 10.1021/bi00195a017. [DOI] [PubMed] [Google Scholar]
  21. Hedstrom L., Szilagyi L., Rutter W. J. Converting trypsin to chymotrypsin: the role of surface loops. Science. 1992 Mar 6;255(5049):1249–1253. doi: 10.1126/science.1546324. [DOI] [PubMed] [Google Scholar]
  22. Hedstrom L. Trypsin: a case study in the structural determinants of enzyme specificity. Biol Chem. 1996 Jul-Aug;377(7-8):465–470. [PubMed] [Google Scholar]
  23. Jackson C. M., Nemerson Y. Blood coagulation. Annu Rev Biochem. 1980;49:765–811. doi: 10.1146/annurev.bi.49.070180.004001. [DOI] [PubMed] [Google Scholar]
  24. Krishnaswamy S., Church W. R., Nesheim M. E., Mann K. G. Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism. J Biol Chem. 1987 Mar 5;262(7):3291–3299. [PubMed] [Google Scholar]
  25. Kurachi K., Davie E. W. Isolation and characterization of a cDNA coding for human factor IX. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6461–6464. doi: 10.1073/pnas.79.21.6461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lawson J. H., Mann K. G. Cooperative activation of human factor IX by the human extrinsic pathway of blood coagulation. J Biol Chem. 1991 Jun 15;266(17):11317–11327. [PubMed] [Google Scholar]
  27. Lenting P. J., ter Maat H., Clijsters P. P., Donath M. J., van Mourik J. A., Mertens K. Cleavage at arginine 145 in human blood coagulation factor IX converts the zymogen into a factor VIII binding enzyme. J Biol Chem. 1995 Jun 23;270(25):14884–14890. doi: 10.1074/jbc.270.25.14884. [DOI] [PubMed] [Google Scholar]
  28. LiCata V. J., Ackers G. K. Long-range, small magnitude nonadditivity of mutational effects in proteins. Biochemistry. 1995 Mar 14;34(10):3133–3139. doi: 10.1021/bi00010a001. [DOI] [PubMed] [Google Scholar]
  29. Lindquist P. A., Fujikawa K., Davie E. W. Activation of bovine factor IX (Christmas factor) by factor XIa (activated plasma thromboplastin antecedent) and a protease from Russell's viper venom. J Biol Chem. 1978 Mar 25;253(6):1902–1909. [PubMed] [Google Scholar]
  30. Lottenberg R., Christensen U., Jackson C. M., Coleman P. L. Assay of coagulation proteases using peptide chromogenic and fluorogenic substrates. Methods Enzymol. 1981;80(Pt 100):341–361. doi: 10.1016/s0076-6879(81)80030-4. [DOI] [PubMed] [Google Scholar]
  31. Lozier J. N., Monroe D. M., Stanfield-Oakley S., Lin S. W., Smith K. J., Roberts H. R., High K. A. Factor IX New London: substitution of proline for glutamine at position 50 causes severe hemophilia B. Blood. 1990 Mar 1;75(5):1097–1104. [PubMed] [Google Scholar]
  32. MARES-GUIA M., SHAW E. STUDIES ON THE ACTIVE CENTER OF TRYPSIN. THE BINDING OF AMIDINES AND GUANIDINES AS MODELS OF THE SUBSTRATE SIDE CHAIN. J Biol Chem. 1965 Apr;240:1579–1585. [PubMed] [Google Scholar]
  33. Mather T., Oganessyan V., Hof P., Huber R., Foundling S., Esmon C., Bode W. The 2.8 A crystal structure of Gla-domainless activated protein C. EMBO J. 1996 Dec 16;15(24):6822–6831. [PMC free article] [PubMed] [Google Scholar]
  34. McRae B. J., Kurachi K., Heimark R. L., Fujikawa K., Davie E. W., Powers J. C. Mapping the active sites of bovine thrombin, factor IXa, factor Xa, factor XIa, factor XIIa, plasma kallikrein, and trypsin with amino acid and peptide thioesters: development of new sensitive substrates. Biochemistry. 1981 Dec 8;20(25):7196–7206. doi: 10.1021/bi00528a022. [DOI] [PubMed] [Google Scholar]
  35. Padmanabhan K., Padmanabhan K. P., Tulinsky A., Park C. H., Bode W., Huber R., Blankenship D. T., Cardin A. D., Kisiel W. Structure of human des(1-45) factor Xa at 2.2 A resolution. J Mol Biol. 1993 Aug 5;232(3):947–966. doi: 10.1006/jmbi.1993.1441. [DOI] [PubMed] [Google Scholar]
  36. Perona J. J., Craik C. S. Structural basis of substrate specificity in the serine proteases. Protein Sci. 1995 Mar;4(3):337–360. doi: 10.1002/pro.5560040301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rezaie A. R., Neuenschwander P. F., Morrissey J. H., Esmon C. T. Analysis of the functions of the first epidermal growth factor-like domain of factor X. J Biol Chem. 1993 Apr 15;268(11):8176–8180. [PubMed] [Google Scholar]
  38. Rezaie A. R. Role of residue 99 at the S2 subsite of factor Xa and activated protein C in enzyme specificity. J Biol Chem. 1996 Sep 27;271(39):23807–23814. doi: 10.1074/jbc.271.39.23807. [DOI] [PubMed] [Google Scholar]
  39. Roberts H. R. Molecular biology of hemophilia B. Thromb Haemost. 1993 Jul 1;70(1):1–9. [PubMed] [Google Scholar]
  40. Stubbs M. T., Huber R., Bode W. Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin. FEBS Lett. 1995 Nov 13;375(1-2):103–107. doi: 10.1016/0014-5793(95)01190-p. [DOI] [PubMed] [Google Scholar]
  41. Wells J. A. Additivity of mutational effects in proteins. Biochemistry. 1990 Sep 18;29(37):8509–8517. doi: 10.1021/bi00489a001. [DOI] [PubMed] [Google Scholar]
  42. van Dieijen G., Tans G., Rosing J., Hemker H. C. The role of phospholipid and factor VIIIa in the activation of bovine factor X. J Biol Chem. 1981 Apr 10;256(7):3433–3442. [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES