Structure-function relationships of a catalytically efficient beta-D-xylosidase.
Beta-D-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (kcat, kcat/Km) for promoting hydrolysis of 1,4-beta-D-xylooligosaccharides. 1H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl- beta-D-xylopyranoside (4NPX) and 1,4-beta-D-xylobiose (X2). Progress curves of 4-nitrophenyl-beta-D-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of kcat and kcat/Km decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites -1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites beyond -1 are blocked by protein and subsites beyond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode kcat and kcat/Km by <103 and to a similar extent for substrates 4NPX and 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With 4NPX and 4NPA, pH governs kcat/Km with pKa values of 5.0 and 7.0 assigned to D14 and E186, respectively. kcat(4NPX) has a pKa value of 7.0 and kcat(4NPA) is pH independent above pH 4.0, suggesting that the catalytically inactive, "dianionic" enzyme form (D14-E187-) binds 4NPX but not 4NPA.
Jordan
Douglas B
DB
US Department of Agriculture, Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, Peoria, IL 61604, USA. jordand@ncaur.usda.gov
Li
Xin-Liang
XL
Dunlap
Christopher A
CA
Whitehead
Terence R
TR
Cotta
Michael A
MA
D002384:Catalysis; D004789:Enzyme Activation; D004795:Enzyme Stability; D020357:Selenomonas / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry* / Q000648:ultrastructure*
D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase