Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1990 Aug 15;270(1):91–96. doi: 10.1042/bj2700091

Essential carboxy groups in xylanase A.

M R Bray 1, A J Clarke 1
PMCID: PMC1131682  PMID: 2396996

Abstract

An endo-1,4-beta-xylanase of Schizophyllum commune was purified to homogeneity through a modified procedure employing DEAE-Sepharose CL-6B and gel-filtration chromatography on Sephadex G-50. The role of carboxy groups in the catalytic mechanism was delineated through chemical modification studies. The water-soluble carbodi-imide 1-(4-azonia-4,4-dimethylpentyl)-3-ethylcarbodi-imide iodide (EAC) inactivated the xylanase rapidly and completely in a pseudo-first-order process. Other carbodi-imides and Woodward's Reagent K were less effective in decreasing enzymic activity. Significant protection of the enzyme against EAC inactivation was provided by a mixture of neutral xylo-oligomers. The pH-dependence of the EAC inactivation revealed the presence of a critical ionizable group with a pKa value of 6.6 in the active site of the xylanase. Treatment of the enzyme with diethyl pyrocarbonate resulted in modification of all three histidine residues in the enzyme with 100% retention of original enzymic activity. Titration of the enzyme with 5,5-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetimide and p-chloromercuribenzoate indicated the absence of free/reactive thiol groups. Reaction of the xylanase with tetranitromethane did not result in a significant activity loss as a result of modification of tyrosine residues.

Full text

PDF
91

Selected References

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

  1. Blumenkrantz N., Asboe-Hansen G. New method for quantitative determination of uronic acids. Anal Biochem. 1973 Aug;54(2):484–489. doi: 10.1016/0003-2697(73)90377-1. [DOI] [PubMed] [Google Scholar]
  2. Carraway K. L., Koshland D. E., Jr Reaction of tyrosine residues in proteins with carbodiimide reagents. Biochim Biophys Acta. 1968 Jun 26;160(2):272–274. doi: 10.1016/0005-2795(68)90102-5. [DOI] [PubMed] [Google Scholar]
  3. Chan V. W., Jorgensen A. M., Borders C. L., Jr Inactivation of bovine thrombin by water-soluble carbodiimides: the essential carboxyl group has a pKa of 5.51. Biochem Biophys Res Commun. 1988 Mar 15;151(2):709–716. doi: 10.1016/s0006-291x(88)80338-3. [DOI] [PubMed] [Google Scholar]
  4. Chipman D. M., Sharon N. Mechanism of lysozyme action. Science. 1969 Aug 1;165(3892):454–465. doi: 10.1126/science.165.3892.454. [DOI] [PubMed] [Google Scholar]
  5. Clarke A. J., Yaguchi M. The role of carboxyl groups in the function of endo-beta-1,4-glucanase from Schizophyllum commune. Eur J Biochem. 1985 Jun 3;149(2):233–238. doi: 10.1111/j.1432-1033.1985.tb08917.x. [DOI] [PubMed] [Google Scholar]
  6. Dekker R. F., Richards G. N. Hemicellulases: their occurrence, purification, properties, and mode of action. Adv Carbohydr Chem Biochem. 1976;32:277–352. doi: 10.1016/s0065-2318(08)60339-x. [DOI] [PubMed] [Google Scholar]
  7. ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
  8. George A. L., Jr, Borders C. L., Jr Essential carboxyl residues in yeast enolase. Biochem Biophys Res Commun. 1979 Mar 15;87(1):59–65. doi: 10.1016/0006-291x(79)91646-2. [DOI] [PubMed] [Google Scholar]
  9. Hurst P. L., Sullivan P. A., Shepherd M. G. Chemical modification of cellulase from Aspergillus niger. Biochem J. 1977 Dec 1;167(3):549–556. doi: 10.1042/bj1670549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Høj P. B., Rodriguez E. B., Stick R. V., Stone B. A. Differences in active site structure in a family of beta-glucan endohydrolases deduced from the kinetics of inactivation by epoxyalkyl beta-oligoglucosides. J Biol Chem. 1989 Mar 25;264(9):4939–4947. [PubMed] [Google Scholar]
  11. Inokuchi N., Iwama M., Takahashi T., Irie M. Modification of a glucoamylase from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide. J Biochem. 1982 Jan;91(1):125–133. doi: 10.1093/oxfordjournals.jbchem.a133669. [DOI] [PubMed] [Google Scholar]
  12. Kersters-Hilderson H., Van Doorslaer E., Lippens M., De Bruyne C. K. The pH dependence and group modification of beta-D-xylosidase from Bacillus pumilus: evidence for sulfhydryl and histidyl groups. Arch Biochem Biophys. 1984 Oct;234(1):61–72. doi: 10.1016/0003-9861(84)90324-2. [DOI] [PubMed] [Google Scholar]
  13. Keskar S. S., Srinivasan M. C., Deshpande V. V. Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site. Biochem J. 1989 Jul 1;261(1):49–55. doi: 10.1042/bj2610049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kochhar S., Dua R. D. An active-site carboxyl group in liquefying alpha-amylase: specific chemical modification. Biosci Rep. 1984 Jul;4(7):613–619. doi: 10.1007/BF01121919. [DOI] [PubMed] [Google Scholar]
  15. LEVY H. M., LEBER P. D., RYAN E. M. INACTIVATION OF MYOSIN BY 2,4-DINITROPHENOL AND PROTECTION BY ADENOSINE TRIPHOSPHATE AND OTHER PHOSPHATE COMPOUNDS. J Biol Chem. 1963 Nov;238:3654–3659. [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. Moranelli F., Barbier J. R., Dove M. J., MacKay R. M., Seligy V. L., Yaguchi M., Willick G. E. A clone coding for Schizophyllum commune beta-glucosidase: homology with a yeast beta-glucosidase. Biochem Int. 1986 Jun;12(6):905–912. [PubMed] [Google Scholar]
  18. Mühlrad A., Hegyi G., Horányi M. Studies on the properties of chemically modified actin. 3. Carbethoxylation. Biochim Biophys Acta. 1969 May;181(1):184–190. doi: 10.1016/0005-2795(69)90240-2. [DOI] [PubMed] [Google Scholar]
  19. Paice M. G., Jurasek L., Carpenter M. R., Smillie L. B. Production, characterization, and partial amino acid sequence of xylanase A from Schizophyllum commune. Appl Environ Microbiol. 1978 Dec;36(6):802–808. doi: 10.1128/aem.36.6.802-808.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Scrutton M. C., Utter M. F. Pyruvate carboxylase. V. Interaction of the enzyme with adenosine triphosphate. J Biol Chem. 1965 Oct;240(10):3714–3723. [PubMed] [Google Scholar]
  21. Sokolovsky M., Riordan J. F., Vallee B. L. Tetranitromethane. A reagent for the nitration of tyrosyl residues in proteins. Biochemistry. 1966 Nov;5(11):3582–3589. doi: 10.1021/bi00875a029. [DOI] [PubMed] [Google Scholar]
  22. TIMELL T. E. WOOD HEMICELLULOSES. I. Adv Carbohydr Chem. 1964;19:247–302. [PubMed] [Google Scholar]
  23. Timkovich R. Detection of the stable addition of carbodiimide to proteins. Anal Biochem. 1977 May 1;79(1-2):135–143. doi: 10.1016/0003-2697(77)90387-6. [DOI] [PubMed] [Google Scholar]
  24. Yaguchi M., Roy C., Rollin C. F., Paice M. G., Jurasek L. A fungal cellulase shows sequence homology with the active site of hen egg-white lysozyme. Biochem Biophys Res Commun. 1983 Oct 31;116(2):408–411. doi: 10.1016/0006-291x(83)90537-5. [DOI] [PubMed] [Google Scholar]

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

RESOURCES