B-D-Xylosidase from Selenomonas ruminantium:Catalyzed Reactions with Natural and Artificial Substrates

摘要

Catalytically efficient (3-D-xylosidase from Selenomonas ruminantium (SXA)exhibits pKas 5 and 7 (assigned to catalytic base, D14, and catalytic acid, E186) for k_(cat)/K_mwith substrates 1,4-(3-D-xylobiose (X2) and 1,4-β-D-xylotriose (X3). Catalytically inactive,dianionic SXA (D14-E186-) has threefold lower affinity than catalytically active,monoanionic SXA (D14-E186H) for X2 and X3, whereas D14-E186- has twofold higheraffinity than D14-E186~Hfor 4-nitrophenyl-β-D-xylopyranoside (4NPX), and D14-E186-has no affinity for 4-nitrophenyl-α-L-arabinofuranoside. Anomeric isomers, α-D-xylose and(3-D-xylose, have similar affinity for SXA. 4-Nitrophenol competitively inhibits SXA-catalyzed hydrolysis of 4NPX. SXA steady-state kinetic parameters account for completeprogress curves of SXA-catalyzed hydrolysis reactions.