beta-D-Xylosidase from Selenomonas ruminantium: role of glutamate 186 in catalysis revealed by site-directed mutagenesis, alternate substrates, and active-site inhibitor

Applied Biochemistry and Biotechnology
Douglas B Jordan, Jay D Braker

Abstract

beta-D-Xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D-xylose. Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic base (D14, pKa 5.0) and catalytic acid (E186, pKa 7.2). Biphasic inhibition by triethanolamine of E186A preparations reveals minor contamination by wild-type-like enzyme, the contaminant likely originating from translational misreading. Titration of E186A preparations with triethanolamine allows resolution of binding and kinetic parameters of the E186A mutant from those of the contaminant. The E186A mutation abolishes the pKa assigned to E186; mutant enzyme binds only the neutral aminoalcohol pH-independent K(triethanolamine)(i)=19 mM), whereas wild-type enzyme binds only the cationic aminoalcohol pH-independent K(triethanolamine)(i)=0.065 mM. At pH 7.0 and 25 degrees C, relative kinetic parameter, k(4NPX)(cat)=k(4NPA)(cat), for substrates 4-nitrophenyl-beta-D-xylopyranoside (4NPX) and 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA) of E186A is 100-fold that of wild-type enzyme, consistent with the view that, on the enzyme, protonation is of...Continue Reading

References

Nov 1, 1989·Analytical Biochemistry·S C Gill, P H von Hippel
Nov 5, 1997·Current Opinion in Structural Biology·B Henrissat, G Davies
Apr 17, 2003·Journal of Industrial Microbiology & Biotechnology·Badal C Saha
Jul 2, 2003·Current Opinion in Microbiology·Dalia Shallom, Yuval Shoham
Mar 9, 2006·Current Opinion in Chemical Biology·Kevin A GrayMark Emptage
Jul 13, 2007·Applied Biochemistry and Biotechnology·Douglas B JordanMichael A Cotta
May 15, 2008·Applied Biochemistry and Biotechnology·Douglas B JordanMichael A Cotta
Oct 28, 2008·Applied Biochemistry and Biotechnology·Douglas B Jordan, Jay D Braker
Nov 1, 2008·Biochimica Et Biophysica Acta·Douglas B JordanJay D Braker

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Citations

Oct 12, 2012·Applied Microbiology and Biotechnology·Douglas B JordanJay D Braker
Apr 30, 2011·Journal of Industrial Microbiology & Biotechnology·Douglas B JordanChamroeun Heng

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