Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Jan 1;321(Pt 1):83–88. doi: 10.1042/bj3210083

Protease digestion studies of an equilibrium intermediate in the unfolding of creatine kinase.

T Webb 1, P J Jackson 1, G E Morris 1
PMCID: PMC1218039  PMID: 9003404

Abstract

Protease digestion experiments have been used to characterize the structure of an equilibrium intermediate in the unfolding of creatine kinase (CK) by low concentrations (0.625 M) of guanidine hydrochloride (GdnHCl). Eighteen of the major products of digestion by trypsin, chymotrypsin and endoproteinase Glu-C have been identified by microsequencing after separation by SDS/PAGE and electroblotting on poly(vinylidene difluoride) membranes. The C-terminal portion (Gly215 to Lys380) was much more resistant to digestion than the N-terminal portion (Pro1 to Gly133), although the area most sensitive to proteolysis was in the middle of the CK sequence (Arg134 to Arg214). These experiments are consistent with the two-domain model for the CK monomer. The structure of the intermediate is proposed to consist of a folded C-terminal domain and a partly folded N-terminal domain separated by an unfolded central linker. Protease susceptibility is clustered within two N-terminal regions and one central region. These regions are evidently exposed as a result of the partial unfolding and/or separation of the N-terminal domain. Further evidence for the structure of this intermediate comes from gel filtration studies. Treatment of CK with 0.625 M GdnHCl resulted in slow aggregation at 37 degrees C, but not at 12 degrees C, a phenomenon previously reported for phosphoglycerate kinase. The aggregation did not occur at higher GdnHCl concentrations and was unaffected by a reducing agent. It is proposed that aggregation is a consequence of non-specific interactions between hydrophobic regions, possibly domain/domain interfaces, which become exposed in the intermediate.

Full Text

The Full Text of this article is available as a PDF (448.3 KB).

Selected References

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

  1. Banks R. D., Blake C. C., Evans P. R., Haser R., Rice D. W., Hardy G. W., Merrett M., Phillips A. W. Sequence, structure and activity of phosphoglycerate kinase: a possible hinge-bending enzyme. Nature. 1979 Jun 28;279(5716):773–777. doi: 10.1038/279773a0. [DOI] [PubMed] [Google Scholar]
  2. Bickerstaff G. F., Price N. C. Evidence for active subunits of matrix-bound creatine kinase. FEBS Lett. 1976 May 1;64(2):319–322. doi: 10.1016/0014-5793(76)80319-5. [DOI] [PubMed] [Google Scholar]
  3. Couthon F., Clottes E., Ebel C., Vial C. Reversible dissociation and unfolding of dimeric creatine kinase isoenzyme MM in guanidine hydrochloride and urea. Eur J Biochem. 1995 Nov 15;234(1):160–170. doi: 10.1111/j.1432-1033.1995.160_c.x. [DOI] [PubMed] [Google Scholar]
  4. Eppenberger H. M., Dawson D. M., Kaplan N. O. The comparative enzymology of creatine kinases. I. Isolation and characterization from chicken and rabbit tissues. J Biol Chem. 1967 Jan 25;242(2):204–209. [PubMed] [Google Scholar]
  5. Fontana A., Fassina G., Vita C., Dalzoppo D., Zamai M., Zambonin M. Correlation between sites of limited proteolysis and segmental mobility in thermolysin. Biochemistry. 1986 Apr 22;25(8):1847–1851. doi: 10.1021/bi00356a001. [DOI] [PubMed] [Google Scholar]
  6. Fritz-Wolf K., Schnyder T., Wallimann T., Kabsch W. Structure of mitochondrial creatine kinase. Nature. 1996 May 23;381(6580):341–345. doi: 10.1038/381341a0. [DOI] [PubMed] [Google Scholar]
  7. Furter R., Furter-Graves E. M., Wallimann T. Creatine kinase: the reactive cysteine is required for synergism but is nonessential for catalysis. Biochemistry. 1993 Jul 13;32(27):7022–7029. doi: 10.1021/bi00078a030. [DOI] [PubMed] [Google Scholar]
  8. Gross M., Furter-Graves E. M., Wallimann T., Eppenberger H. M., Furter R. The tryptophan residues of mitochondrial creatine kinase: roles of Trp-223, Trp-206, and Trp-264 in active-site and quaternary structure formation. Protein Sci. 1994 Jul;3(7):1058–1068. doi: 10.1002/pro.5560030708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gross M., Lustig A., Wallimann T., Furter R. Multiple-state equilibrium unfolding of guanidino kinases. Biochemistry. 1995 Aug 22;34(33):10350–10357. doi: 10.1021/bi00033a005. [DOI] [PubMed] [Google Scholar]
  10. Gross M., Wyss M., Furter-Graves E. M., Wallimann T., Furter R. Reconstitution of active octameric mitochondrial creatine kinase from two genetically engineered fragments. Protein Sci. 1996 Feb;5(2):320–330. doi: 10.1002/pro.5560050216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grossman S. H. An equilibrium study of the dependence of secondary and tertiary structure of creatine kinase on subunit association. Biochim Biophys Acta. 1994 Nov 16;1209(1):19–23. doi: 10.1016/0167-4838(94)90131-7. [DOI] [PubMed] [Google Scholar]
  12. Grossman S. H., Mixon D. Further characterization of the reassembly of creatine kinase and effect of substrate. Arch Biochem Biophys. 1985 Feb 1;236(2):797–806. doi: 10.1016/0003-9861(85)90686-1. [DOI] [PubMed] [Google Scholar]
  13. Grossman S. H., Pyle J., Steiner R. J. Kinetic evidence for active monomers during the reassembly of denatured creatine kinase. Biochemistry. 1981 Oct 13;20(21):6122–6128. doi: 10.1021/bi00524a032. [DOI] [PubMed] [Google Scholar]
  14. Haas R. C., Korenfeld C., Zhang Z. F., Perryman B., Roman D., Strauss A. W. Isolation and characterization of the gene and cDNA encoding human mitochondrial creatine kinase. J Biol Chem. 1989 Feb 15;264(5):2890–2897. [PubMed] [Google Scholar]
  15. Hubbard S. J., Eisenmenger F., Thornton J. M. Modeling studies of the change in conformation required for cleavage of limited proteolytic sites. Protein Sci. 1994 May;3(5):757–768. doi: 10.1002/pro.5560030505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lin L., Perryman M. B., Friedman D., Roberts R., Ma T. S. Determination of the catalytic site of creatine kinase by site-directed mutagenesis. Biochim Biophys Acta. 1994 May 18;1206(1):97–104. doi: 10.1016/0167-4838(94)90077-9. [DOI] [PubMed] [Google Scholar]
  18. Morris G. E., Cartwright A. J. Monoclonal antibody studies suggest a catalytic site at the interface between domains in creatine kinase. Biochim Biophys Acta. 1990 Jul 6;1039(3):318–322. doi: 10.1016/0167-4838(90)90265-h. [DOI] [PubMed] [Google Scholar]
  19. Morris G. E., Frost L. C., Newport P. A., Hudson N. Monoclonal antibody studies of creatine kinase. Antibody-binding sites in the N-terminal region of creatine kinase and effects of antibody on enzyme refolding. Biochem J. 1987 Nov 15;248(1):53–59. doi: 10.1042/bj2480053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Morris G. E., Jackson P. J. Identification by protein microsequencing of a proteinase-V8-cleavage site in a folding intermediate of chick muscle creatine kinase. Biochem J. 1991 Dec 15;280(Pt 3):809–811. doi: 10.1042/bj2800809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Morris G. E., Man N. T. Changes at the N-terminus of human brain creatine kinase during a transition between inactive folding intermediate and active enzyme. Biochim Biophys Acta. 1992 Apr 8;1120(2):233–238. doi: 10.1016/0167-4838(92)90276-j. [DOI] [PubMed] [Google Scholar]
  22. Morris G. E. Monoclonal antibody studies of creatine kinase. The ART epitope: evidence for an intermediate in protein folding. Biochem J. 1989 Jan 15;257(2):461–469. doi: 10.1042/bj2570461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mühlebach S. M., Gross M., Wirz T., Wallimann T., Perriard J. C., Wyss M. Sequence homology and structure predictions of the creatine kinase isoenzymes. Mol Cell Biochem. 1994 Apr-May;133-134:245–262. doi: 10.1007/BF01267958. [DOI] [PubMed] [Google Scholar]
  24. Nguyen thi Man, Cartwright A. J., Osborne M., Morris G. E. Structural changes in the C-terminal region of human brain creatine kinase studied with monoclonal antibodies. Biochim Biophys Acta. 1991 Jan 29;1076(2):245–251. doi: 10.1016/0167-4838(91)90274-4. [DOI] [PubMed] [Google Scholar]
  25. PORTER R. R. The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. Biochem J. 1959 Sep;73:119–126. doi: 10.1042/bj0730119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Webb T., Jackson P. J., Morris G. E. Probing protein structure with proteases: studies of an equilibrium intermediate in protein unfolding. Biochem Soc Trans. 1995 Aug;23(3):477S–477S. doi: 10.1042/bst023477s. [DOI] [PubMed] [Google Scholar]
  27. White T. K., Wilson J. E. Isolation and characterization of the discrete N- and C-terminal halves of rat brain hexokinase: retention of full catalytic activity in the isolated C-terminal half. Arch Biochem Biophys. 1989 Nov 1;274(2):375–393. doi: 10.1016/0003-9861(89)90451-7. [DOI] [PubMed] [Google Scholar]
  28. Williamson J., Greene J., Chérif S., Milner-White E. J. Heterogeneity of rabbit muscle creatine kinase and limited proteolysis by proteinase K. Biochem J. 1977 Dec 1;167(3):731–737. doi: 10.1042/bj1670731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wilson J. E. The use of monoclonal antibodies and limited proteolysis in elucidation of structure-function relationships in proteins. Methods Biochem Anal. 1991;35:207–250. doi: 10.1002/9780470110560.ch4. [DOI] [PubMed] [Google Scholar]
  30. Wyss M., James P., Schlegel J., Wallimann T. Limited proteolysis of creatine kinase. Implications for three-dimensional structure and for conformational substrates. Biochemistry. 1993 Oct 12;32(40):10727–10735. doi: 10.1021/bi00091a025. [DOI] [PubMed] [Google Scholar]
  31. Zhou H. M., Zhang X. H., Yin Y., Tsou C. L. Conformational changes at the active site of creatine kinase at low concentrations of guanidinium chloride. Biochem J. 1993 Apr 1;291(Pt 1):103–107. doi: 10.1042/bj2910103. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES