Structure-Function Relationships of a Catalytically Efficient β-D-Xylosidase.

Douglas B. Jordan; Xin-Liang Li; Christopher A. Dunlap; Terence R. Whitehead; Michael A. Cotta

β-D-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (kcat, kcat/Km) for promoting hydrolysis of 1,4-β-D-xylooligosaccharides.1H nuclear magnetic resonance experiments indicate the family 43 glycosidehydrolase acts through an inversion mechanism on substrates 4-nitrophenyl-β-D-xylopyranoside (4NPX) and 1,4-β-D-xylobiose (X2). Progress curves of4-nitrophenyl-β-D-xylobioside, xylotetraose and xylohexaose reactionsindicate that one residue from the nonreducing end of substrate is cleavedper catalytic cycle without processivity. Values of kcat and kcat/Km decrease forxylooligosaccharides longer than X2, illustrating the importance to catalysisof subsites -1 and 1 and the lack there of subsite 2. Homology models ofthe enzyme active site with docked substrates show that subsites beyond -1 areblocked by protein and subsites beyond 1 are not formed; they suggest thatD14 and E186 serve catalysis as general base and general acid, respectively.Individual mutations, D14A and E186A, erode kcat and kcat/Km by <103 and toa similar extent for substrates 4NPX and 4-nitrophenyl-α-L-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With4NPX and 4NPA, pH governs kcat/Km with pKa values of 5.0 and 7.0 assignedto 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.

SURFACE chemistry; PHYSICAL & theoretical chemistry; MAGNETIC resonance; HYDROGEN-ion concentration

Applied Biochemistry & Biotechnology, 2007, Vol 141, Issue 1, p51

0273-2289

Academic Journal

10.1007/s12010-007-9210-8