Structure of the two-subsite β-d-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino]propane

Joseph S. Brunzelle, Douglas B. Jordan*, Darrell R. McCaslin, Andrzej Olczak, Zdzislaw Wawrzak

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

43 Scopus citations

Abstract

The three-dimensional structure of the catalytically efficient β-xylosidase from Selenomonas ruminantium in complex with competitive inhibitor 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP) was determined by using X-ray crystallography (1.3 Å resolution). Most H bonds between inhibitor and protein occur within subsite -1, including one between the carboxyl group of E186 and an N group of BTP. The other N of BTP occupies subsite +1 near K99. E186 (pKa 7.2) serves as catalytic acid. The pH (6-10) profile for 1 / Ki(BTP) is bell-shaped with pKa's 6.8 and 7.8 on the acidic limb assigned to E186 and inhibitor groups and 9.9 on the basic limb assigned to inhibitor. Mutation K99A eliminates pKa 7.8, strongly suggesting that the BTP monocation binds to the dianionic enzyme D14-E186-. A sedimentation equilibrium experiment estimates a Kd ([dimer]2/[tetramer]) of 7 × 10-9 M. Similar kcat and kcat/Km values were determined when the tetramer/dimer ratio changes from 0.0028 to 26 suggesting that dimers and tetramers are equally active forms.

Original languageEnglish (US)
Pages (from-to)157-166
Number of pages10
JournalArchives of biochemistry and biophysics
Volume474
Issue number1
DOIs
StatePublished - Jun 1 2008

Keywords

  • GH43
  • Glycoside hydrolase
  • Protein crystallography
  • Sedimentation equilibrium
  • Structure-function
  • α-l-Arabinofuranosidase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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