title: Aminoalcohols as probes of the two-subsite active site of beta-D-xylosidase from Selenomonas ruminantium.
abstract: 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(i)(triethanolamine) 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-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.
authors.affiliation: National Center for Agricultural Utilization Research, U.S. Department of Agriculture, Agricultural Research Service, Peoria, IL 61604, USA. douglas.jordan@ars.usda.gov
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Jordan
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Mertens
authors.forename: Jay D
authors.initials: JD
authors.lastname: Braker
mesh_terms: D000605:Amino Alcohols / Q000737:chemistry / Q000494:pharmacology*; D001089:Arabinose / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004983:Ethanolamines; D006026:Glycoside Hydrolases / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D020357:Selenomonas / Q000201:enzymology*; D013379:Substrate Specificity; D014995:Xylosidases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D000605:Amino Alcohols; D004791:Enzyme Inhibitors; D004983:Ethanolamines; D006027:Glycosides; C008646:4-nitrophenyl beta-D-xyloside; C017567:4-nitrophenyl-alpha-L-arabinofuranoside; C009546:triethanolamine; C020283:diethanolamine; D001089:Arabinose; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase