Aminoalcohols as probes of the two-subsite active site of β-d-xylosidase from Selenomonas ruminantium

Catalysis and inhibitor binding by the GH43 β-xylosidase are governed by the protonation states of catalytic base (D14, pKa 5.0) and catalytic acid (E186, pKa 7.2) which reside in subsite − 1 of the two-subsite active site. Cationic aminoalcohols are shown to bind exclusively to subsite − 1 of the catalytically-inactive, dianionic enzyme (D14−E186−). Enzyme (E) and aminoalcohols (A) form E–A with the affinity progression: triethanolamine > diethanolamine > ethanolamine. E186A mutation raises the Kitriethanolamine 1000-fold. By occupying subsite − 1 with aminoalcohols, affinity of monosaccharide inhibitors (I) for subsite + 1 is demonstrated. The single access route for ligands into the active site dictates ordered formation of E–A followed by E–A–I. E–A–I forms with the affinity progression: ethanolamine > diethanolamine > triethanolamine. The latter affinity progression is seen in formation of E–A–substrate complexes with substrate 4-nitrophenyl-β-d-xylopyranoside (4NPX). Inhibition patterns of aminoalcohols versus 4NPX appear competitive, noncompetitive, and uncompetitive depending on the strength of E–A–4NPX. E–A–substrate complexes form weakly with substrate 4-nitrophenyl-α-l-arabinofuranoside (4NPA), and inhibition patterns appear competitive. Biphasic inhibition by triethanolamine reveals minor (< 0.03%) contamination of E186A preparations (including a His-Tagged form) by wild-type-like enzyme, likely originating from translational misreading. Aminoalcohols are useful in probing glycoside hydrolases; they cause artifacts when used unwarily as buffer components.