Structure-function relationships of a catalytically efficient β-D-xylosidase

beta-D-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (k(cat), k(cat)/K-m ) for promoting hydrolysis of 1,4-beta-D-xylooligosaccharides. H-1 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 k(cat) and k(cat) / K-m 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 k(cat) and k(cat)/K-m by < 10(3) 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 k(cat) / K-m with pK(a) values of 5.0 and 7.0 assigned to D14 and E186, respectively. k(cat) (4NPX) has a pK(a) value of 7.0 and k(cat) (4NPA) is pH independent above pH 4.0, suggesting that the catalytically inactive, "dianionic" enzyme form (DA(-)E187(-)) binds 4NPX but not 4NPA.