Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1984 Sep 15;222(3):769–776. doi: 10.1042/bj2220769

Proteinase from germinating bean cotyledons. Evidence for involvement of a thiol group in catalysis.

C Csoma, L Polgár
PMCID: PMC1144241  PMID: 6385962

Abstract

To degrade storage proteins germinating seeds synthesize proteinases de novo that can be inhibited by thiol-blocking reagents [Baumgartner & Chrispeels (1977) Eur. J. Biochem. 77, 223-233]. We have elaborated a procedure for isolation of such a proteinase from the cotyledons of Phaseolus vulgaris. The purification procedure involved fractionation of the cotyledon homogenate with acetone and with (NH4)2SO4 and successive chromatographies on DEAE-cellulose, activated thiol-Sepharose Sepharose and Sephacryl S-200. The purified enzyme has an Mr of 23,400, proved to be highly specific for the asparagine side chain and blocking of its thiol group resulted in loss of the catalytic activity. The chemical properties of the thiol group of the bean enzyme were investigated by acylation with t-butyloxycarbonyl-L-asparagine p-nitro-phenyl ester and by alkylations with iodoacetamide and iodoacetate. Deviations from normal pH-rate profile were observed, which indicated that the thiol group is not a simple functional group, but constitutes a part of an interactive system at the active site. The pKa value for acylation and the magnitude of the rate constant for alkylation with iodoacetate revealed that the bean proteinase possesses some properties not shared by papain and the other cysteine proteinases studied to date.

Full text

PDF
770

Images in this article

773Fig. 2.
on p.773

Selected References

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

  1. BENDER M. L., KEZDY J. MECHANISM OF ACTION OF PROTEOLYTIC ENZYMES. Annu Rev Biochem. 1965;34:49–76. doi: 10.1146/annurev.bi.34.070165.000405. [DOI] [PubMed] [Google Scholar]
  2. Bai Y., Hayashi R. Properties of the single sulfhydryl group of carboxypeptidase Y. Effects of alkyl and aromatic mercurials on activities toward various synthetic substrates. J Biol Chem. 1979 Sep 10;254(17):8473–8479. [PubMed] [Google Scholar]
  3. Baumgartner B., Chrispeels M. J. Purification and characterization of vicilin peptidohydrolase, the major endopeptidase in the cotyledons of mung-bean seedlings. Eur J Biochem. 1977 Jul 15;77(2):223–233. doi: 10.1111/j.1432-1033.1977.tb11661.x. [DOI] [PubMed] [Google Scholar]
  4. Brocklehurst K., Mushiri S. M., Patel G., Willenbrock F. Evidence for a close similarity in the catalytic sites of papain and ficin in near-neutral media despite differences in acidic and alkaline media. Kinetics of the reactions of papain and ficin with chloroacetate. Biochem J. 1982 Jan 1;201(1):101–104. doi: 10.1042/bj2010101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brocklehurst K. Specific covalent modification of thiols: applications in the study of enzymes and other biomolecules. Int J Biochem. 1979;10(4):259–274. doi: 10.1016/0020-711x(79)90088-0. [DOI] [PubMed] [Google Scholar]
  6. Chrispeels M. J., Boulter D. Control of storage protein metabolism in the cotyledons of germinating mung beans: role of endopeptidase. Plant Physiol. 1975 Jun;55(6):1031–1037. doi: 10.1104/pp.55.6.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  8. Halász P., Polgár L. Negatively charged reactants as probes in the study of the essential mercaptide-imidazolium ion-pair of thiolenzymes. Eur J Biochem. 1977 Oct 3;79(2):491–494. doi: 10.1111/j.1432-1033.1977.tb11832.x. [DOI] [PubMed] [Google Scholar]
  9. Hazen G. G., Hause J. A., Hubicki J. A. An automated system for the quantitative determination of proteolytic enzymes using azocasein. Ann N Y Acad Sci. 1965 Nov 9;130(2):761–768. doi: 10.1111/j.1749-6632.1965.tb12620.x. [DOI] [PubMed] [Google Scholar]
  10. Polgár L., Halász P. Current problems in mechanistic studies of serine and cysteine proteinases. Biochem J. 1982 Oct 1;207(1):1–10. doi: 10.1042/bj2070001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Polgár L. Ion-pair formation as a source of enhanced reactivity of the essential thiol group of D-glyceraldehyde-3-phosphate dehydrogenase. Eur J Biochem. 1975 Feb 3;51(1):63–71. doi: 10.1111/j.1432-1033.1975.tb03907.x. [DOI] [PubMed] [Google Scholar]
  12. Polgár L. On the mode of activation of the catalytically essential sulfhydryl group of papain. Eur J Biochem. 1973 Feb 15;33(1):104–109. doi: 10.1111/j.1432-1033.1973.tb02660.x. [DOI] [PubMed] [Google Scholar]
  13. Yomo H., Srinivasan K. Protein Breakdown and Formation of Protease in Attached and Detached Cotyledons of Phaseolus vulgaris L. Plant Physiol. 1973 Dec;52(6):671–673. doi: 10.1104/pp.52.6.671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Yomo H., Varner J. E. Control of the formation of amylases and proteases in the cotyledons of germinating peas. Plant Physiol. 1973 Apr;51(4):708–713. doi: 10.1104/pp.51.4.708. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES