Submitted to: Archives Of Biochemistry and Biophysics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/22/2007
Publication Date: 6/8/2007
Citation: Jordan, D.B., Braker, J.D. 2007. Inhibition of the two-subsite beta-d-xylosidase from Selenomonas ruminantium by sugars: competitive, noncompetitive, double binding, and slow binding modes. Archives of Biochemistry and Biophysics. 465(1):231-246. Interpretive Summary: Agricultural biomass like crop residues, grain processing byproducts, dedicated energy crops (e.g., switchgrass), etc., represent an abundant, renewable feedstock for production of ethanol and other valuable products if practical conversion technologies can be developed. These materials are rich in complex carbohydrates that must first be broken down to simple sugars that can be fermented by microorganisms to ethanol and other products. A critical step in the development of new conversion processes is the discovery and development of new enzymes to convert these complex materials to simple sugars. We have discovered an enzyme involved in the final step in the hydrolysis of xylan, the second most abundant carbohydrate in plants. This enzyme produces the simple sugar, xylose, more efficiently than other enzymes described by other workers. The enzyme reaction is inhibited by monosaccharides, and the inhibitor binding modes are defined here. Our results will help us and other researchers in the development of new bioconversion strategies to produce fuel ethanol economically.
Technical Abstract: The active site of the GH43 beta-xylosidase from Selenomonas ruminantium comprises two subsites and single access route for ligands. Inhibition of enzyme-catalyzed hydrolysis of 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA) and 4-nitrophenyl-beta-D-xylopyranoside (4NPX) by sugars was analyzed according to a two subsite binding scheme. Steady-state kinetic experiments that included enzyme (E), inhibitors (I and X), and substrates 4NPA and 4NPX establish formation of EI (all inhibitors), EII (double binding of D-arabinose, L-arabinose, D-erythrose, and D-ribose), EIX (simultaneous binding by mixed inhibitor pairs L-arabinose/D-ribose, L-arabinose/D-xylose, and D-erythrose/D-ribose), and EIS (simultaneous binding by inhibitor/substrate pairs D-xylose/4NPX, L-arabinose/4NPA, L-arabinose/4NPX, L-xylose/4NPX, and L-xylose/4NPA). pH dependencies of inhibition were analyzed according to a diprotic model governed by pKa's 5 and 7, assigned to catalytic base D14 and catalytic acid E186. Of the four inhibitors in the pH study, none bind to the diprotonated D14**HE186**H enzyme; D14**-E186**H binds D-ribose with 1.6-fold the affinity of D14**-E186**-, and D14**-E186**- binds D-xylose, D-glucose, and L-arabinose with 1.7-, 3.1-, and 2.6-fold the affinity of D14**-E186**H. EII, EIX, and EIS occur only with the D14**-E186**H enzyme. It is speculated that the negative charge of D14**-E186**- repels sugar moieties from binding fully in subsite -1 so they must occupy a portion of subsite +1 which prevents binding of a second ligand to subsite +1. At pH 7, binding of two equivalents of L-arabinose to the D14**-E186**H enzyme is separated by equilibrium Ki values (first equivalent binds with 6-fold the affinity of the second) and kinetically (the first equivalent binds rapidly and the second equivalent binds slowly).