beta-D-Xylosidase from Selenomonas ruminantium: role of glutamate 186 in catalysis revealed by site-directed mutagenesis, alternate substrates, and active-site inhibitor.
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 greater importance to the transition state of 4NPA whereas ring deformation dominates the transition state of 4NPX.
Jordan
Douglas Brian
DB
Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 N. University Street, Peoria, IL 61604, USA. douglas.jordan@ars.usda.gov
Braker
Jay D
JD
D002384:Catalysis; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D020357:Selenomonas / Q000201:enzymology*; D013379:Substrate Specificity; D014995:Xylosidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D018698:Glutamic Acid; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase