Aminoalcohols as probes of the two-subsite active site Of beta-D-xylosidase from Selenomonas ruminantium

作者：Jordan Douglas B^*; Mertens Jeffrey A; Braker Jay D

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2009, 1794(1): 144-158.

DOI：10.1016/j.bbapap.2008.09.015

• alpha-L-Arabinofuranosidase
• Glycoside hydrolase
• Noncompetitive inhibition
• Structure function
• Translational missense

Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic base (D14, pK(a) 5.0) and catalytic acid (E186, pK(a) 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 K vertical bar(riethanolamine) 1000-fold. By occupying subsite -1 with aminoalcohols, affinity of monosaccharide inhibitors (1) 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-1. E-A-1 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-beta-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-alpha-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. Published by Elsevier B.V.

• 出版日期2009-1