β-d-Xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-Directed Mutagenesis, Alternate Substrates, and Active-Site Inhibitor

Abstract

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.

Engineering lower inhibitor affinities in β-d-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145

“…Active-site mutations expressing k cat 4NPX/4NPA values lower than wildtype SXA have been located in subsite -1 surrounding the glycone, while mutations effecting k cat 4NPX/4NPA values higher than that of the wild type are located in subsite ?1 surrounding the aglycone. Mutations that produce higher k cat 4NPX/4NPA ratios than that of wild-type SXA, such as reported here for position 145 variants, identify native residues as promoting other events of the single transitionstate reaction mechanism conducted by SXA such as protonation of the aglycone leaving group or C1 migration [22]. The k cat 4NPX/4NPA ratio of W145G is 2.5-fold that of wild-type SXA and was supporting evidence in conjunction with subsite-specific inhibitor binding studies for previous assignment of the residue's influence on K i D-xylose and K i D-glucose as through subsite ?1 [7], with the present k cat 4NPX/4NPA results further substantiating the conclusion that the position 145 mutations influence subsite ?1 in changing catalytic events.…”

“…Substrates, buffers, and other reagents [7,17,21,22], circular dichroism spectroscopy [17], HPLC with pulsed amperometric detection [19], isothermal calorimetry [21], SDS-PAGE analysis [15], UV-V is spectroscopy [17], and molecular modeling [7] have been described. D-Xylobiose (X2) was obtained from Wako Chemicals USA (Richmond, VA).…”

“…Because X2 contains the energetically more difficult xylosyl glycone in common with 4NPX and X2, which obviously facilitates SXA-catalyzed hydrolysis of X2 (xylosyl aglycone) over 4NPX (4-nitrophenol aglycone) for wild-type SXA as the k cat X2/4NPX value is 6.4 for wild type, it might be expected that mutations that lower k cat X2/ 4NPX are associated with facilitating protonation of the aglycone leaving group, as found for the E186A mutant [22], or C1 migration events. Indeed the W145G mutant expresses k cat X2/4NPX 3.30, half that of the wild-type SXA, suggesting that the mutation impacts subsite ?1 of SXA.…”

“…The latter experiments relied on our earlier work that shows amino alcohols bind exclusively to subsite -1 and that a lower ratio k cat 4NPX /k cat 4NPA (4NPA, 4-nitrophenyl a-L-arabinofuranoside; Fig. 1) is associated with influences on subsite -1 and a higher ratio to influences on subsite ?1 [17,21,22]. W145G exhibits K i values for D-glucose and D-xylose that are twofold and threefold those of wild-type SXA, respectively.…”

“…SXA has the simplest active site possible for an efficient glycoside hydrolase by comprising only two subsites [3], subsite -1 for binding the substrate glycone and subsite ?1 for binding the substrate aglycone. Because the simplicity of SXA lends itself to study, and because of the potential practical importance of SXA, a good deal has been learned about its structure-function properties [3,[16][17][18][19][20][21][22][23].…”