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. 1981 Jan 1;193(1):55–65. doi: 10.1042/bj1930055

Inactivation of aspartyl proteinases by butane-2,3-dione. Modification of tryptophan and tyrosine residues and evidence against reaction of arginine residues.

J C Gripon, T Hofmann
PMCID: PMC1162575  PMID: 6796042

Abstract

Butane-2,3-dione inactivates the aspartyl proteinases from Penicillium roqueforti and Penicillium caseicolum, as well as pig pepsin, penicillopepsin and Rhizopus pepsin, at pH 6.0 in the presence of light but not in the dark. The inactivation is due to a photosensitized modification of tryptophan and tyrosine residues. In the dark none of the amino acid residues, not even arginine residues, is modified even after several days. In the light one arginine residue in pig pepsin is lost at a rate that is comparable with the rate of inactivation; however, the loss of the single arginine residue in the aspartyl proteinase of P. roqueforti and the second arginine residue of pig pepsin is slower than the loss of activity; penicillopepsin is devoid of arginine. Loss of most of the activity is accompanied by the following amino acid losses: P. roqueforti aspartyl proteinase, about two tryptophan and six tyrosine residues; penicillopepsin, about two tryptophan and three tyrosine residues; pig pepsin, about four tryptophan and most of the tyrosine residues. Modification of histidine residues was too slow to contribute to inactivation. None of the other residues, including half-cystine and methionine residues (when present), was modified even after prolonged incubation. The inactivation of P. roqueforti aspartyl proteinase and pig pepsin appears due to non-specific modification of several residues. With penicillopepsin, however, the reaction is more limited and initially affects only those tryptophan and tyrosine residues that lie in the active-site groove. In the presence of pepstatin the rate of inactivation is considerably diminished. After prolonged reaction a general structural breakdown occurs.

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Selected References

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

  1. Cunningham A., Wang H. M., Jones S. R., Chiericato G., Rao L., Harris C. I., Rhee S. H., Hofmann T. Amino acid sequence of penicillopepsin. IV. Myxobacter AL-1 protease II and Staphylococcus aureus protease fragments and homology with pig pepsin and chymosin. Can J Biochem. 1976 Oct;54(10):902–914. doi: 10.1139/o76-128. [DOI] [PubMed] [Google Scholar]
  2. Fliss H., Viswanatha T. 2,3-butanedione as a photosensitizing agent: application to alpha-amino acids and alpha-chymotrypsin. Can J Biochem. 1979 Nov;57(11):1267–1272. doi: 10.1139/o79-168. [DOI] [PubMed] [Google Scholar]
  3. Fruton J. S. The mechanism of the catalytic action of pepsin and related acid proteinases. Adv Enzymol Relat Areas Mol Biol. 1976;44:1–36. doi: 10.1002/9780470122891.ch1. [DOI] [PubMed] [Google Scholar]
  4. GREEN N. M., WITKOP B. OXIDATION STUDIES OF INDOLES AND THE TERTIARY STRUCTURE OF PROTEINS. Trans N Y Acad Sci. 1964 Apr;26:659–669. doi: 10.1111/j.2164-0947.1964.tb01933.x. [DOI] [PubMed] [Google Scholar]
  5. Gennari G., Jori G. Acetone-sensitized anaerobic photo-oxidation of methionine. FEBS Lett. 1970 Sep 24;10(2):129–131. doi: 10.1016/0014-5793(70)80433-1. [DOI] [PubMed] [Google Scholar]
  6. Gripon J. C. Inactivation of Penicillium roqueforti acid protease by specific pepsin inhibitors. Biochimie. 1976;58(6):747–749. doi: 10.1016/s0300-9084(76)80401-4. [DOI] [PubMed] [Google Scholar]
  7. Gripon J. C., Rhee S. H., Hofmann T. N-terminal amino acid sequences of acid proteases: acid proteases from Penicillium roqueforti and Rhizopus chinensis and alignment with penicillopepsin and mammalian proteases. Can J Biochem. 1977 May;55(5):504–506. doi: 10.1139/o77-071. [DOI] [PubMed] [Google Scholar]
  8. Hofmann T. Penicillopepsin. Methods Enzymol. 1976;45:434–452. doi: 10.1016/s0076-6879(76)45038-3. [DOI] [PubMed] [Google Scholar]
  9. Hsu I. N., Delbaere L. T., James M. N., Hofmann T. Penicillopepsin from Penicillium janthinellum crystal structure at 2.8 A and sequence homology with porcine pepsin. Nature. 1977 Mar 10;266(5598):140–145. doi: 10.1038/266140a0. [DOI] [PubMed] [Google Scholar]
  10. Huang W. Y., Tang J. Modification of an arginyl residue in pepsin by 2,3-butanedione. J Biol Chem. 1972 May 10;247(9):2704–2710. [PubMed] [Google Scholar]
  11. Irvine G. B., Elmore D. T. A radiochemical titrant for the determination of the operational molarity of solutions of acid proteinases. Biochem J. 1979 Nov 1;183(2):389–394. doi: 10.1042/bj1830389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. James M. N., Hsu I. N., Delbaere L. T. Mechanism of acid protease catalysis based on the crystal structure of penicillopepsin. Nature. 1977 Jun 30;267(5614):808–813. doi: 10.1038/267808a0. [DOI] [PubMed] [Google Scholar]
  13. Kitson T. M., Knowles J. R. The effect of arginine modification on the pH dependence of pepsin activity. FEBS Lett. 1971 Sep 1;16(4):337–338. doi: 10.1016/0014-5793(71)80384-8. [DOI] [PubMed] [Google Scholar]
  14. Nakatani H., Kitagishi K., Hiromi K. Spectroscopic studies of pepsin and its complex with Streptomyces pepsin inhibitor. Biochim Biophys Acta. 1976 Dec 8;452(2):521–524. doi: 10.1016/0005-2744(76)90203-5. [DOI] [PubMed] [Google Scholar]
  15. Penke B., Ferenczi R., Kovács K. A new acid hydrolysis method for determining tryptophan in peptides and proteins. Anal Biochem. 1974 Jul;60(1):45–50. doi: 10.1016/0003-2697(74)90129-8. [DOI] [PubMed] [Google Scholar]
  16. Riordan J. F. Arginyl residues and anion binding sites in proteins. Mol Cell Biochem. 1979 Jul 31;26(2):71–92. doi: 10.1007/BF00232886. [DOI] [PubMed] [Google Scholar]
  17. Strickland E. H. Aromatic contributions to circular dichroism spectra of proteins. CRC Crit Rev Biochem. 1974 Jan;2(1):113–175. doi: 10.3109/10409237409105445. [DOI] [PubMed] [Google Scholar]
  18. Tang J., Sepulveda P., Marciniszyn J., Jr, Chen K. C., Huang W. Y., Tao N., Liu D., Lanier J. P. Amino-acid sequence of porcine pepsin. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3437–3439. doi: 10.1073/pnas.70.12.3437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wang T. T., Dorrington K. J., Hofmann T. Activation of the action of penicillopepsin on leucyl-tyrosyl-amide by a non-substrate peptide and evidence for a conformational change associated with a secondary binding site. Biochem Biophys Res Commun. 1974 Apr 8;57(3):865–869. doi: 10.1016/0006-291x(74)90626-3. [DOI] [PubMed] [Google Scholar]
  20. Zevaco C., Hermier J., Gripon J. C. Le système protéolytique de Penicillium roqueforti. 2. Purification et propriétés de la protéase acide. Biochimie. 1973;55(11):1353–1360. [PubMed] [Google Scholar]

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