Investigation of the active site of the extracellular β-D-xylosidase from Aspergillus carbonarius

Tünde Kiss, Anikó Erdei, L. Kiss

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

The catalytic amino acid residues of the extracellular β-D-xylosidase (β-D-xyloside xylohydrolase, EC 3.2. 1.37) from Aspergillus carbonarius was investigated by the pH dependence of reaction kinetic parameters and chemical modifications of the enzyme. The pH dependence curves gave apparent pK values of 2.7 and 6.4 for the free enzyme, while pK value of 4.0 was obtained for the enzyme-substrate complex using p-nitrophenyl β-D-xyloside as a substrate. These results suggested that a carboxylate group and a protonated group - presumably a histidine residue - took part in the binding of the substrate but only a carboxylate group was essential in the substrate cleavage. Carbodiimide- and Woodward's reagent K-mediated chemical modifications of the enzyme also supported that a carboxylate residue, located in the active center, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.4, proving that a carboxylate residue relevant for hydrolysis was modified. During modification Vmax decreased to 10% of that of the unmodified enzyme and Km remained unchanged, supporting that the modified carboxylate group participated in the cleavage and not in the binding of the substrate. We synthesized and tested a new, potential affinity label, N-bromoacetyl-β-D-xylopyranosylamine for β-D-xylosidase. The A. carbonarius β-D-xylosidase was irreversible inactivated by N-bromoacetyl-β-D-xylopyranosylamine. The competitive inhibitor β-D-xylopyranosyl azide protected the enzyme from inactivation proving that the inactivation took place in the active center. Kinetic analysis indicated that one molecule of reagent was necessary for inactivation of one molecule of the enzyme.

Original languageEnglish
Pages (from-to)188-194
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume399
Issue number2
DOIs
Publication statusPublished - Mar 15 2002

Fingerprint

Xylosidases
Aspergillus
Catalytic Domain
Enzymes
Substrates
Chemical modification
Affinity Labels
Carbodiimides
Molecules
Azides
Catalysis
Kinetic parameters
Histidine
Reaction kinetics
Hydrolysis
Amino Acids
Kinetics

Keywords

  • β-D-xylosidase
  • Affinity label
  • Aspergillus carbonarius
  • Chemical modification
  • N-bromoacetyl-β-D-xylopyranosylamine

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Investigation of the active site of the extracellular β-D-xylosidase from Aspergillus carbonarius. / Kiss, Tünde; Erdei, Anikó; Kiss, L.

In: Archives of Biochemistry and Biophysics, Vol. 399, No. 2, 15.03.2002, p. 188-194.

Research output: Contribution to journalArticle

@article{e74c924e34134550a72a19488b30fc28,
title = "Investigation of the active site of the extracellular β-D-xylosidase from Aspergillus carbonarius",
abstract = "The catalytic amino acid residues of the extracellular β-D-xylosidase (β-D-xyloside xylohydrolase, EC 3.2. 1.37) from Aspergillus carbonarius was investigated by the pH dependence of reaction kinetic parameters and chemical modifications of the enzyme. The pH dependence curves gave apparent pK values of 2.7 and 6.4 for the free enzyme, while pK value of 4.0 was obtained for the enzyme-substrate complex using p-nitrophenyl β-D-xyloside as a substrate. These results suggested that a carboxylate group and a protonated group - presumably a histidine residue - took part in the binding of the substrate but only a carboxylate group was essential in the substrate cleavage. Carbodiimide- and Woodward's reagent K-mediated chemical modifications of the enzyme also supported that a carboxylate residue, located in the active center, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.4, proving that a carboxylate residue relevant for hydrolysis was modified. During modification Vmax decreased to 10{\%} of that of the unmodified enzyme and Km remained unchanged, supporting that the modified carboxylate group participated in the cleavage and not in the binding of the substrate. We synthesized and tested a new, potential affinity label, N-bromoacetyl-β-D-xylopyranosylamine for β-D-xylosidase. The A. carbonarius β-D-xylosidase was irreversible inactivated by N-bromoacetyl-β-D-xylopyranosylamine. The competitive inhibitor β-D-xylopyranosyl azide protected the enzyme from inactivation proving that the inactivation took place in the active center. Kinetic analysis indicated that one molecule of reagent was necessary for inactivation of one molecule of the enzyme.",
keywords = "β-D-xylosidase, Affinity label, Aspergillus carbonarius, Chemical modification, N-bromoacetyl-β-D-xylopyranosylamine",
author = "T{\"u}nde Kiss and Anik{\'o} Erdei and L. Kiss",
year = "2002",
month = "3",
day = "15",
doi = "10.1006/abbi.2002.2753",
language = "English",
volume = "399",
pages = "188--194",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Investigation of the active site of the extracellular β-D-xylosidase from Aspergillus carbonarius

AU - Kiss, Tünde

AU - Erdei, Anikó

AU - Kiss, L.

PY - 2002/3/15

Y1 - 2002/3/15

N2 - The catalytic amino acid residues of the extracellular β-D-xylosidase (β-D-xyloside xylohydrolase, EC 3.2. 1.37) from Aspergillus carbonarius was investigated by the pH dependence of reaction kinetic parameters and chemical modifications of the enzyme. The pH dependence curves gave apparent pK values of 2.7 and 6.4 for the free enzyme, while pK value of 4.0 was obtained for the enzyme-substrate complex using p-nitrophenyl β-D-xyloside as a substrate. These results suggested that a carboxylate group and a protonated group - presumably a histidine residue - took part in the binding of the substrate but only a carboxylate group was essential in the substrate cleavage. Carbodiimide- and Woodward's reagent K-mediated chemical modifications of the enzyme also supported that a carboxylate residue, located in the active center, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.4, proving that a carboxylate residue relevant for hydrolysis was modified. During modification Vmax decreased to 10% of that of the unmodified enzyme and Km remained unchanged, supporting that the modified carboxylate group participated in the cleavage and not in the binding of the substrate. We synthesized and tested a new, potential affinity label, N-bromoacetyl-β-D-xylopyranosylamine for β-D-xylosidase. The A. carbonarius β-D-xylosidase was irreversible inactivated by N-bromoacetyl-β-D-xylopyranosylamine. The competitive inhibitor β-D-xylopyranosyl azide protected the enzyme from inactivation proving that the inactivation took place in the active center. Kinetic analysis indicated that one molecule of reagent was necessary for inactivation of one molecule of the enzyme.

AB - The catalytic amino acid residues of the extracellular β-D-xylosidase (β-D-xyloside xylohydrolase, EC 3.2. 1.37) from Aspergillus carbonarius was investigated by the pH dependence of reaction kinetic parameters and chemical modifications of the enzyme. The pH dependence curves gave apparent pK values of 2.7 and 6.4 for the free enzyme, while pK value of 4.0 was obtained for the enzyme-substrate complex using p-nitrophenyl β-D-xyloside as a substrate. These results suggested that a carboxylate group and a protonated group - presumably a histidine residue - took part in the binding of the substrate but only a carboxylate group was essential in the substrate cleavage. Carbodiimide- and Woodward's reagent K-mediated chemical modifications of the enzyme also supported that a carboxylate residue, located in the active center, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.4, proving that a carboxylate residue relevant for hydrolysis was modified. During modification Vmax decreased to 10% of that of the unmodified enzyme and Km remained unchanged, supporting that the modified carboxylate group participated in the cleavage and not in the binding of the substrate. We synthesized and tested a new, potential affinity label, N-bromoacetyl-β-D-xylopyranosylamine for β-D-xylosidase. The A. carbonarius β-D-xylosidase was irreversible inactivated by N-bromoacetyl-β-D-xylopyranosylamine. The competitive inhibitor β-D-xylopyranosyl azide protected the enzyme from inactivation proving that the inactivation took place in the active center. Kinetic analysis indicated that one molecule of reagent was necessary for inactivation of one molecule of the enzyme.

KW - β-D-xylosidase

KW - Affinity label

KW - Aspergillus carbonarius

KW - Chemical modification

KW - N-bromoacetyl-β-D-xylopyranosylamine

UR - http://www.scopus.com/inward/record.url?scp=0037088547&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037088547&partnerID=8YFLogxK

U2 - 10.1006/abbi.2002.2753

DO - 10.1006/abbi.2002.2753

M3 - Article

VL - 399

SP - 188

EP - 194

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 2

ER -