Skip to main content
SpringerLink
Log in

The Crystal Structure of a Trypsin-like Mutant Chymotrypsin: The Role of Position 226 in the Activity and Specificity of S189D Chymotrypsin

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

The crystal structure of the S189D+A226G rat chymotrypsin-B mutant has been determined at 2.2 Å resolution. This mutant is the most trypsin-like mutant so far in the line of chymotrypsin-to-trypsin conversions, aiming for a more complete understanding of the structural basis of substrate specificity in pancreatic serine proteases. A226G caused significant rearrangements relative to S189D chymotrypsin, allowing an internal conformation of Asp189 which is close to that in trypsin. Serious distortions remain, however, in the activation domain, including zymogen-like features. The pH-profile of activity suggests that the conformation of the S1–site of the mutant is influenced also by the P1 residue of the substrate.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

AMC:

Amino-4-Methyl Coumarin

BPTI:

Bovine pancreatic trypsin inhibitor

CABS:

4-(Cyclohexylamino)-1-butanesulfonic acid

CCP4:

Collaborative Computational Project Number 4

CHES:

2-(Cyclohexylamino)ethanesulfonic acid

DPI:

Dispersion precision indicator

ESRF:

European Synchrotron Radiation Facility

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

MES:

2-(N-Morpholino)ethanesulfonic acid

MOPS:

3-(N-Morpholino)propanesulfonic acid

MUGB:

4-Guanidinobenzoic acid 4-methylumbelliferyl ester hydrochloride

PDB:

Protein data bank

PEG:

Polyethylene glycol

SBTI:

Soybean trypsin inhibitor

SDS-PAGE:

Sodium dodecyl sulfate - polyacrylamide gel electrophoresis

TLS:

Translational, librational, screw

TRIS:

Tris(hydroxymethyl)aminomethane

References

  1. Schechter I, Berger A (1967) Biochem Biophys Res Commun 27:157–162

    Article  CAS  Google Scholar 

  2. Gráf L, Jancsó A, Szilágyi L, Hegyi Gy, Pintér K, Náray-Szabó G, Hepp J, Medzihradszky K, Rutter WJ (1988) Proc Natl Acad Sci USA 85:4961–4965

    Article  Google Scholar 

  3. Hedstrom L, Szilágyi L, Rutter WJ (1992) Science 255:1249–1253

    Article  CAS  Google Scholar 

  4. Hedstrom L, Perona JJ, Rutter WJ (1994) Biochemistry 33:8757–8763

    Article  CAS  Google Scholar 

  5. Hedstrom L, Farr Jones S, Kettner CA, Rutter WJ (1994) Biochemistry 33:8764–8769

    Article  CAS  Google Scholar 

  6. Gráf L (1995) In: Zwilling R (ed) Natural sciences and human thought. Springer-Verlag, Berlin, Heidelberg, pp 139–148

  7. Steitz TA, Henderson R, Blow DM (1969) J Mol Biol 46:337–348

    Article  CAS  Google Scholar 

  8. Venekei I, Szilágyi L, Gráf L, Rutter WJ (1996) FEBS Lett 379:143–147

    Article  CAS  Google Scholar 

  9. Craik CS, Largman C, Fletcher T, Roczniak S, Barr PJ, Fletterick RJ, Rutter WJ (1985) Science 228:291–297

    Article  CAS  Google Scholar 

  10. Wilke ME, Higaki JN, Craik CS, Fletterick RJ (1991) J Mol Biol 219:525–532

    Article  CAS  Google Scholar 

  11. Jelinek B, Antal J, Venekei I, Gráf L (2004) Protein Eng Des Sel 17:127–131

    Article  CAS  Google Scholar 

  12. Jameson GW, Adams DV, Kyle WS, Elmore DT (1973) Biochem J 131:107–117

    CAS  Google Scholar 

  13. CCP 4 (1994) Acta Cryst D 50:760–763

    Google Scholar 

  14. Navaza J (2001) Acta Crystallogr D Biol Crystallogr 57:1367–1372

    Article  CAS  Google Scholar 

  15. Szabó E, Venekei I, Böcskei Zs, Náray-Szabó G, Gráf L (2003) J Mol Biol 331(5):1121–1130

    Article  Google Scholar 

  16. Terwilliger TC (2000) Acta Cryst D56:965–972

    CAS  Google Scholar 

  17. Emsley P, Cowtan K (2004) Acta Cryst D60:2126–2132

    CAS  Google Scholar 

  18. Murshudov GN, Vagin AA, Dodson EJ (1997) Acta Cryst D53:240–255

    CAS  Google Scholar 

  19. Hooft RW, Vriend G, Sander C, Abola EE (1996) Nature 381:272

    Article  CAS  Google Scholar 

  20. Laskowski RA, MAcArtur MW, Moss DS, Thornton JM (1993) J Appl Crystallog 26:283–291

    Article  CAS  Google Scholar 

  21. Luzzati PV (1952) Acta Cryst 5:802–810

    Article  Google Scholar 

  22. Cruickshank DWJ (1999) Acta Cryst D55:583–601

    CAS  Google Scholar 

  23. Lesk AM (1991) Protein architecture: a practical guide. IRL Press, Oxford

    Google Scholar 

  24. Madsen D, Kleywegt GJ (2002) J Appl Crystallog 35:137–139

    Article  CAS  Google Scholar 

  25. Marquart M, Walter J, Deisenhofer J, Bode W, Huber R (1983) Acta Crystallogr Sect B v39:480

    Article  Google Scholar 

  26. Brady K, Wei AZ, Ringe D, Abeles RH (1990) Biochemistry v29:7600–7607

    Article  Google Scholar 

  27. Segel IH (1993) Enzyme Kinetics: Behavior and analysis of rapid equilibrium and steady-state enzyme systems, Wiley Classics Library Edition

  28. Dementiev A, Dobó J, Gettins PGW (2006) J Biol Chem 281(6):3452–3457

    Article  CAS  Google Scholar 

  29. Katz B, Kossiakoff A (1986) J Biol Chem 261(33):15480-15485

    CAS  Google Scholar 

  30. Fodor K, Harmat V, Neutze R, Szilágyi L, Gráf L, Katona G (2006) Biochemistry 21;45(7):2114–2121

    Article  Google Scholar 

  31. Radisky ES, Lee JM, Lu CJ, Koshland DE Jr (2006) Proc Natl Acad Sci U S A 103(18):6835–6840

    Article  CAS  Google Scholar 

  32. Fuhrmann CN, Daugherty MD, Agard DA (2006) J Am Chem Soc 128(28):9086–9102

    Article  CAS  Google Scholar 

  33. Liu B, Schofield CJ, Wilmouth RC (2006) J Biol Chem 281(33):24024–24035

    Article  CAS  Google Scholar 

  34. Fersht AR (1972) J Mol Biol 64(2):497–509

    Article  CAS  Google Scholar 

  35. Verheyden G, Matrai J, Volckaert G, Engelborghs Y (2004) Prot Sci 13:2533–2540

    Article  CAS  Google Scholar 

  36. Szabó E, Böcskei Zs, Náray-Szabó G, Gráf L (1999) Eur J Biochem 263(1):20–26

    Article  Google Scholar 

  37. Bode W, Schwager P, Huber R (1978) J Mol Biol 118:99–112

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful for the beamline staff at the ESRF ID14–2 beamline for their expert assistance. This work was supported by the Hungarian Research Fund OTKA TS049812 and T047154 to L.G., G. K. acknowledges the support from EMBO and CEA.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Eötvös Loránd University, Pázmány s. 1/C, Budapest, 1117, Hungary

    Balázs Jelinek, István Venekei & László Gráf

  2. Institut de Biologie Structurale, UMR 5075, CEA/CNRS/UJF, 41 avenue Jules Horowitz, 38027, Grenoble Cedex 1, France

    Gergely Katona

  3. Biotechnology Research Group, Hungarian Academy of Sciences, Pázmány s. 1/C, Budapest, 1117, Hungary

    Krisztián Fodor & László Gráf

Authors
  1. Balázs Jelinek
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gergely Katona
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Krisztián Fodor
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. István Venekei
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. László Gráf
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Balázs Jelinek.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jelinek, B., Katona, G., Fodor, K. et al. The Crystal Structure of a Trypsin-like Mutant Chymotrypsin: The Role of Position 226 in the Activity and Specificity of S189D Chymotrypsin. Protein J 27, 79–87 (2008). https://doi.org/10.1007/s10930-007-9110-3

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-007-9110-3

Keywords

Search

Navigation