Isolation and divalent-metal activation of a ß-xylosidase, RUM630-BX.

The gene encoding RUM630-BX, a ß-xylosidase/arabinofuranosidase, was identified from activity-based screening of a cow rumen metagenomic library. The recombinant enzyme is activated as much as 14-fold (kcat) by divalent metals Mg(2+), Mn(2+) and Co(2+) but not by Ca(2+), Ni(2+), and Zn(2+). Activation of RUM630-BX by Mg(2+) (t0.5 144 s) is slowed two-fold by prior incubation with substrate, consistent with the X-ray structure of closely related xylosidase RS223-BX that shows the divalent-metal activator is at the back of the active-site pocket so that bound substrate could block its entrance. The enzyme is considerably more active on natural substrates than artificial substrates, with activity (kcat/Km) of 299 s(-1) mM(-1) on xylotetraose being the highest reported.

Rate-limiting steps of a stereochemistry retaining ß-d-xylosidase from Geobacillus stearothermophilus acting on four substrates.

Kinetic experiments of GSXynB2, a GH52 retaining ß-xylosidase, acting on 2-nitrophenyl-ß-d-xylopyranoside (2NPX), 4-nitrophenyl-ß-d-xylopyranoside (4NPX), 4-methylumbelliferyl-ß-d-xylopyranoside (MuX) and xylobiose (X2) were conducted at pH 7.0 and 25 °C. Catalysis proceeds in two steps (xylodidation followed by dexylosidation): E + substrate TO E-xylose + leaving group TO E + xylose. kcat falls into two groups: 4NPX (1.95 s(-1)) and 2NPX, MuX and X2 (15.8 s(-1), 12.6 s(-1), 12.8 s(-1), respectively). Dexylosylation (E-xylose to E + xylose), the common step for the enzymatic hydrolysis of the four substrates, must exceed 15.8 s(-1). kcat of 4NPX would seem mainly limited by xylosylation (step 1) and the other three substrates would seem mainly limited by dexylosylation (step 2) - a conclusion that critically lacks chemical justification (compare 4NPX and 2NPX). Presteady-state rates indicate rapid xylosidation rates for all substrates so a later step (not dexylosidation) is rate-limiting for 4NPX. That 2NPX is an onlier and 4NPX is an outlier (both leaving group pKa of 7.2) of the Brønsted plot pattern (logkcat vs pKa of phenol leaving group) is thus possibly explained by 4NP release. The pH dependency of kcat 2NPX encompasses 2 bell-shaped curves with peaks of pH 3 and pH 7.

X-ray Crystal Structure of Divalent Metal-Activated ß-xylosidase, RS223BX.

We report the X-ray crystal structure of a glycoside hydrolase family 43 ß-xylosidase, RS223BX, which is strongly activated by the addition of divalent metal cations. The 2.69 Å structure reveals that the Ca(2+) cation is located at the back of the active-site pocket. The Ca(2+) is held in the active site by the carboxylate of D85, an "extra" acid residue in comparison to other GH43 active sites. The Ca(2+) is in close contact with a histidine imidazole, which in turn is in contact with the catalytic base (D15) thus providing a mechanism for stabilizing the carboxylate anion of the base and achieve metal activation. The active-site pocket is mirrored by an "inactive-site" pocket of unknown function that resides on the opposite side of the monomer.

Biochemical characterization of uronate dehydrogenases from three Pseudomonads, Chromohalobacter salixigens, and Polaromonas naphthalenivorans.

Enzyme catalysts will be vital in the development of synthetic biology approaches for converting pectinic monosaccharides from citrus and beet processing waste streams to value-added materials. We describe here the biophysical and mechanistic characterization of uronate dehydrogenases from a wide variety of bacterial sources that convert galacturonic acid, the predominate building block of pectin from these plant sources, and glucuronic acid to their corresponding dicarboxylic acids galactarate and glucarate, the latter being a DOE top value biochemical from biomass. The enzymes from Pseudomonas syringae and Polaromonas naphthalenivorans were found to have the highest reported kcat(glucuronic acid) values, on the order of 220-270 s(-1). The thermal stability of this enzyme type is described for the first time here, where it was found that the Kt((0.5)) value range was >20 °C, and the enzyme from Chromohalobacter was moderately thermostable with Kt((0.5))=62.2 °C. The binding mechanism for these bi-substrate enzymes was also investigated in initial rate experiments, where a predominately steady-state ordered binding pattern was indicated.

Directed evolution of GH43 ß-xylosidase XylBH43 thermal stability and L186 saturation mutagenesis.

Directed evolution of ß-xylosidase XylBH43 using a single round of gene shuffling identified three mutations, R45K, M69P, and L186Y, that affect thermal stability parameter K(t)°·5 by -1.8 ± 0.1, 1.7 ± 0.3, and 3.2 ± 0.4 °C, respectively. In addition, a cluster of four mutations near hairpin loop-D83 improved K(t)°·5 by ~3 °C; none of the individual amino acid changes measurably affect K(t)°·5. Saturation mutagenesis of L186 identified the variant L186K as having the most improved K(t)°·5 value, by 8.1 ± 0.3 °C. The L186Y mutation was found to be additive, resulting in K(t)°·5 increasing by up to 8.8 ± 0.3 °C when several beneficial mutations were combined. While k cat of xylobiose and 4-nitrophenyl-ß-D-xylopyranoside were found to be depressed from 8 to 83 % in the thermally improved mutants, K(m), K(ss) (substrate inhibition), and K(i) (product inhibition) values generally increased, resulting in lessened substrate and xylose inhibition.

Rehabilitation of faulty kinetic determinations and misassigned glycoside hydrolase family of retaining mechanism ß-xylosidases.

We obtained Cx1 from a commercial supplier, whose catalog listed it as a ß-xylosidase of glycoside hydrolase family 43. NMR experiments indicate retention of anomeric configuration in its reaction stereochemistry, opposing the assignment of GH43, which follows an inverting mechanism. Partial protein sequencing indicates Cx1 is similar to but not identical to ß-xylosidases of GH52, including Q09LZ0, that have retaining mechanisms. Q09LZ0 ß-xylosidase had been characterized biochemically in kinetic reactions that contained Tris. We overproduced Q09LZ0 and demonstrated that Tris is a competitive inhibitor of the ß-xylosidase. Also, the previous work used grossly incorrect extinction coefficients for product 4-nitrophenol. We redetermined kinetic parameters using reactions that omitted Tris and using correct extinction coefficients for 4-nitrophenol. Cx1 and Q09LZ0 ß-xylosidases were thus shown to possess similar kinetic properties when acting on 4-nitrophenyl-ß-d-xylopyranoside and xylobiose. kcat pH profiles of Cx1 and Q09LZ0 acting on 4-nitrophenyl-ß-d-xylopyranoside and xylobiose have patterns containing two rate increases with increasing acidity, not reported before for glycoside hydrolases. The dexylosylation step of 4-nitrophenyl-ß-d-xylopyranoside hydrolysis mediated by Q09LZ0 is not rate determining for kcat(4NPX).

Activation of a GH43 ß-xylosidase by divalent metal cations: slow binding of divalent metal and high substrate specificity.

RS223-BX of glycoside hydrolase family 43 is a ß-d-xylosidase that is strongly activated (k(cat)/K(m) as much as 116-fold) by the addition of divalent metal cations, Ca(2+), Co(2+), Fe(2+), Mg(2+), Mn(2+) and Ni(2+). Slow activation by Mg(2+) was demonstrated (k(on) 0.013 s(-1) mM(-1), k(off) 0.008 s(-1)) at pH 7.0 and 25 °C. k(off) and k(on) values are independent of Mg(2+) concentration, but k(off) and k(on) are slower in the presence of increasing levels of substrate 4-nitrophenyl-ß-D-xylopyranoside. The kinetics strongly suggest that M(2+) binds to the enzyme rapidly, forming E M(2+), followed by slow isomerization to the activated enzyme, E* M(2+). Moderately high values of kcat (7-30 s(-1)) were found for M(2+)-activated RS223-BX acting on xylobiose (natural substrate) at pH 7.0 and 25 °C. Certain M(2+)-activated RS223-BX exhibit the highest reported values of k(cat)/K(m) of any ß-xylosidase acting on natural substrates: for example, at pH 7.0 and 25°C, xylobiose (Mn(2+), 190 s(-1) mM(-1)), xylotriose (Ca(2+), 150 s(-1) mM(-1)) and xylotetraose (Ca(2+), 260 s(-1) mM(-1)). There is potential for the enzyme to add value to industrial saccharification operations at low substrate and high d-glucose and high d-xylose concentrations.

Divalent metal activation of a GH43 ß-xylosidase.

Depolymerization of xylan, a major fraction of lignocellulosic biomass, releases xylose which can be converted into transportation fuels and chemical feedstocks. A requisite enzyme for the breakdown of xylan is ß-xylosidase. A gene encoding the 324-amino acid ß-xylosidase, RS223-BX, was cloned from an anaerobic mixed microbial culture. This glycoside hydrolase belongs to family 43. Unlike other GH43 enzymes, RS223-BX can be strongly activated by exogenously supplied Ca(2+), Co(2+), Fe(2+), Mg(2+), Mn(2+) and Ni(2+) (e.g., 28-fold by Mg(2+)) and it is inhibited by Cu(2+) or Zn(2+). Sedimentation equilibrium centrifugation experiments indicated that the divalent metal cations mediate multimerization of the enzyme from a dimeric to a tetrameric state, which have equal catalytic activity on an active-site basis. Compared to the determined active sites of other GH43 ß-xylosidases, the predicted active site of RS223-BX contains two additional amino acids with carboxylated side chains that provide potential sites for divalent metal cations to reside. Thus, the divalent metal cations likely occupy the active site and participate in the catalytic mechanism. RS223-BX accepts as substrate xylobiose, arabinobiose, 4-nitrophenyl-ß-D-xylopyranoside, and 4-nitrophenyl-α-L-arabinofuranoside. Additionally, the enzyme has good pH and temperature stabilities and a large K(i) for D-glucose (1.3 M), favorable properties for performance in saccharification reactors.

Highly active ß-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates.

The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. ß-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 is recognized as a source of highly active ß-xylosidases, some of which could have practical applications. The biochemical details of four GH43 ß-xylosidases (those from Alkaliphilus metalliredigens QYMF, Bacillus pumilus, Bacillus subtilis subsp. subtilis str. 168, and Lactobacillus brevis ATCC 367) are examined here. Sedimentation equilibrium experiments indicate that the quaternary states of three of the enzymes are mixtures of monomers and homodimers (B. pumilus) or mixtures of homodimers and homotetramers (B. subtilis and L. brevis). k cat and k cat/K m values of the four enzymes are higher for xylobiose than for xylotriose, suggesting that the enzyme active sites comprise two subsites, as has been demonstrated by the X-ray structures of other GH43 ß-xylosidases. The K i values for D-glucose (83.3-357 mM) and D-xylose (15.6-70.0 mM) of the four enzymes are moderately high. The four enzymes display good temperature (K t (0.5) ∼ 45 °C) and pH stabilities (>4.6 to <10.3). At pH 6.0 and 25 °C, the enzyme from L. brevis ATCC 367 displays the highest reported k cat and k cat/K m on natural substrates xylobiose (407 s(-1), 138 s(-1) mM(-1)), xylotriose (235 s(-1), 80.8 s(-1) mM(-1)), and xylotetraose (146 s(-1), 32.6 s(-1) mM(-1)).

Plant cell walls to ethanol.

Conversion of plant cell walls to ethanol constitutes second generation bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation and separation. Ultimately, it is desirable to combine as many of the biochemical steps as possible in a single organism to achieve CBP (consolidated bioprocessing). A commercially ready CBP organism is currently unreported. Production of second generation bioethanol is hindered by economics, particularly in the cost of pretreatment (including waste management and solvent recovery), the cost of saccharification enzymes (particularly exocellulases and endocellulases displaying kcat ~1 s-1 on crystalline cellulose), and the inefficiency of co-fermentation of 5- and 6-carbon monosaccharides (owing in part to redox cofactor imbalances in Saccharomyces cerevisiae).

Opposing influences by subsite -1 and subsite +1 residues on relative xylopyranosidase/arabinofuranosidase activities of bifunctional ß-D-xylosidase/α-L-arabinofuranosidase.

Conformational inversion occurs 7-8kcal/mol more readily in furanoses than pyranoses. This difference is exploited here to probe for active-site residues involved in distorting pyranosyl substrate toward reactivity. Spontaneous glycoside hydrolysis rates are ordered 4-nitrophenyl-α-l-arabinofuranoside (4NPA)>4-nitrophenyl-ß-d-xylopyranoside (4NPX)>xylobiose (X2). The bifunctional ß-d-xylosidase/α-l-arabinofuranosidase exhibits the opposite order of reactivity, illustrating that the enzyme is well equipped in using pyranosyl groups of natural substrate X2 in facilitating glycoside hydrolysis. Probing the roles of all 17 active-site residues by single-site mutation to alanine and by changing both moieties of substrate demonstrates that the mutations of subsite -1 residues decrease the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support pyranosyl substrate distortion, whereas the mutations of subsite +1 and the subsite -1/+1 interface residues increase the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support other factors, such as C1 migration and protonation of the leaving group. Alanine mutations of subsite -1 residues raise k(cat)(X2/4NPX) and alanine mutations of subsite +1 and interface residues lower k(cat)(X2/4NPX). We propose that pyranosyl substrate distortion is supported entirely by native residues of subsite -1. Other factors leading to the transition state are supported entirely by native residues of subsite +1 and interface residues.

Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural.

An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerevisiae, has been shown to mitigate the toxicity of FFA and HMF by catalyzing the NADPH-dependent conversion to corresponding alcohols, furfuryl alcohol (FFOH) and 5-hydroxymethylfurfuryl alcohol (HMFOH). At pH 7.0 and 25°C, purified Ari1p catalyzes the NADPH-dependent reduction of substrates with the following values (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFA (23.3, 1.82, 12.8), HMF (4.08, 0.173, 23.6), and dl-glyceraldehyde (2.40, 0.0650, 37.0). When acting on HMF and dl-glyceraldehyde, the enzyme operates through an equilibrium ordered kinetic mechanism. In the physiological direction of the reaction, NADPH binds first and NADP(+) dissociates from the enzyme last, demonstrated by k(cat) of HMF and dl-glyceraldehyde that are independent of [NADPH] and (K(ia)(NADPH)/k(cat)) that extrapolate to zero at saturating HMF or dl-glyceraldehyde concentration. Microscopic kinetic parameters were determined for the HMF reaction (HMF+NADPH↔HMFOH+NADP(+)), by applying steady-state, presteady-state, kinetic isotope effects, and dynamic modeling methods. Release of products, HMFOH and NADP(+), is 84% rate limiting to k(cat) in the forward direction. Equilibrium constants, [NADP(+)][FFOH]/[NADPH][FFA][H(+)]=5600×10(7)M(-1) and [NADP(+)][HMFOH]/[NADPH][HMF][H(+)]=4200×10(7)M(-1), favor the physiological direction mirrored by the slowness of hydride transfer in the non-physiological direction, NADP(+)-dependent oxidation of alcohols (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFOH (0.221, 0.00158, 140) and HMFOH (0.0105, 0.000104, 101).

Engineering lower inhibitor affinities in ß-D-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145.

ß-D-Xylosidase/α-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme reported for catalyzing hydrolysis of 1,4-ß-D-xylooligosaccharides to D-xylose. One property that could use improvement is its relatively high affinities for D-glucose and D-xylose (K (i) ~ 10 mM), which would impede its performance as a catalyst in the saccharification of lignocellulosic biomass for the production of biofuels and other value-added products. Previously, we discovered that the W145G variant expresses K(i)(D-glucose) and K(i)(D-xylose) twofold and threefold those of the wild-type enzyme. However, in comparison to the wild type, the variant expresses 11% lower k(cat)(D-xylobiose) and much lower stabilities to temperature and pH. Here, we performed saturation mutagenesis of W145 and discovered that the variants express K (i) values that are 1.5-2.7-fold (D-glucose) and 1.9-4.6-fold (D-xylose) those of wild-type enzyme. W145F, W145L, and W145Y express good stability and, respectively, 11, 6, and 1% higher k(cat)(D-xylobiose) than that of the wild type. At 0.1 M D-xylobiose and 0.1 M D-xylose, kinetic parameters indicate that W145F, W145L, and W145Y catalytic activities are respectively 46, 71, and 48% greater than that of the wild-type enzyme.

Stereochemistry of furfural reduction by a Saccharomyces cerevisiae aldehyde reductase that contributes to in situ furfural detoxification.

Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate-subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by yeast, particularly when the carbon source is acid-treated lignocellulose, which contains furfural at a relatively high concentration. NADPH is Ari1p's best known hydride donor. Here we report the stereochemistry of the hydride transfer step, determined by using (4R)-[4-(2)H]NADPD and (4S)-[4-(2)H]NADPD and unlabeled furfural in Ari1p-catalyzed reactions and following the deuterium atom into products 2-furanmethanol or NADP(+). Analysis of the products demonstrates unambiguously that Ari1p directs hydride transfer from the si face of NADPH to the re face of furfural. The singular orientation of substrates enables construction of a model of the Michaelis complex in the Ari1p active site. The model reveals hydrophobic residues near the furfural binding site that, upon mutation, may increase specificity for furfural and enhance enzyme performance. Using (4S)-[4-(2)H]NADPD and NADPH as substrates, primary deuterium kinetic isotope effects of 2.2 and 2.5 were determined for the steady-state parameters k(cat)(NADPH) and k(cat)/K(m)(NADPH), respectively, indicating that hydride transfer is partially rate limiting to catalysis.

Catalytic properties of two Rhizopus oryzae 99-880 glucoamylase enzymes without starch binding domains expressed in Pichia pastoris.

Catalytic properties of two glucoamylases, AmyC and AmyD, without starch binding domains from Rhizopus oryzae strain 99-880 are determined using heterologously expressed enzyme purified to homogeneity. AmyC and AmyD demonstrate pH optima of 5.5 and 6.0, respectively, nearly one unit higher than the Rhizopus AmyA glucoamylase enzyme. Optimal initial activities are at 60 and 50 °C for AmyC and AmyD, respectively. Inactivation of both enzymes occurs at 50 °C following 30 min pre-incubation. The two enzymes demonstrate substantially slower catalytic rates toward soluble starch relative to AmyA. AmyC has similar k(cat) and K(m) for oligosaccharides to other Rhizopus and Aspergillus glucoamylases; however, the enzyme has a 2-fold lower K(m) (maltose) . AmyD has a 3-fold higher K(m) and lower k(cat) for maltooligosaccharides than AmyC and other glucoamylases. AmyC (but not AmyD) exhibits substrate inhibition. K(i) for substrate inhibition decreases with increasing length of the oligosaccharides. Data from pre-steady-state binding of AmyC to maltose and maltotriose and pre-steady-state to steady-state catalytic turnover experiments of AmyC acting on maltotriose were used to interrogate models of substrate inhibition. In the preferred model, AmyC accumulates an enzyme-maltose-maltotriose dead-end complex in the steady state.

beta-D-Xylosidase from Selenomonas ruminantium: role of glutamate 186 in catalysis revealed by site-directed mutagenesis, alternate substrates, and active-site inhibitor.

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 beta-D-xylosidase.

Beta-D-Xylosidase catalyzes hydrolysis of xylooligosaccharides to D-xylose residues. The enzyme, SXA from Selenomonas ruminantium, is the most active catalyst known for the reaction; however, its activity is inhibited by D-xylose and D-glucose (K (i) values of approximately 10(-2) M). Higher K (i)'s could enhance enzyme performance in lignocellulose saccharification processes for bioethanol production. We report here the development of a two-tier high-throughput screen where the 1 degrees screen selects for activity (active/inactive screen) and the 2 degrees screen selects for a higher K (i(D-xylose)) and its subsequent use in screening approximately 5,900 members of an SXA enzyme library prepared using error-prone PCR. In one variant, termed SXA-C3, K (i(D-xylose)) is threefold and K (i(D-glucose)) is twofold that of wild-type SXA. C3 contains four amino acid mutations, and one of these, W145G, is responsible for most of the lost affinity for the monosaccharides. Experiments that probe the active site with ligands that bind only to subsite -1 or subsite +1 indicate that the changed affinity stems from changed affinity for D-xylose in subsite +1 and not in subsite -1 of the two-subsite active site. Trp145 is 6 A from the active site, and its side chain contacts three active-site residues, two in subsite +1 and one in subsite -1.

Identification of hydrophobic amino acids required for lipid activation of C. elegans CTP:phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CCT), critical for phosphatidylcholine biosynthesis, is activated by translocation to the membrane surface. The lipid activation region of Caenorhabditis elegans CCT is between residues 246 and 266 of the 347 amino acid polypeptide, a region proposed to form an amphipathic alpha helix. When leucine 246, tryptophan 249, isoleucine 256, isoleucine 257, or phenylalanine 260, on the hydrophobic face of the helix, were changed individually to serine low activity was observed in the absence of lipid vesicles, similar to wild-type CCT, while lipid stimulated activity was reduced compared to wild-type CCT. Mutational analysis of phenylalanine 260 implicated this residue as a contributor to auto-inhibition of CCT while mutation of L246, W249, I256, and I257 simultaneously to serine resulted in significantly higher activity in the absence of lipid vesicles and an enzyme that was not lipid activated. These results support a concerted mechanism of lipid activation that requires multiple residues on the hydrophobic face of the putative amphipathic alpha helix.

Aminoalcohols as probes of the two-subsite active site of beta-D-xylosidase from Selenomonas ruminantium.

Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic base (D14, pK(a) 5.0) and catalytic acid (E186, pK(a) 7.2) which reside in subsite -1 of the two-subsite active site. Cationic aminoalcohols are shown to bind exclusively to subsite -1 of the catalytically-inactive, dianionic enzyme (D14(-)E186(-)). Enzyme (E) and aminoalcohols (A) form E-A with the affinity progression: triethanolamine>diethanolamine>ethanolamine. E186A mutation raises the K(i)(triethanolamine) 1000-fold. By occupying subsite -1 with aminoalcohols, affinity of monosaccharide inhibitors (I) for subsite +1 is demonstrated. The single access route for ligands into the active site dictates ordered formation of E-A followed by E-A-I. E-A-I forms with the affinity progression: ethanolamine>diethanolamine>triethanolamine. The latter affinity progression is seen in formation of E-A-substrate complexes with substrate 4-nitrophenyl-beta-d-xylopyranoside (4NPX). Inhibition patterns of aminoalcohols versus 4NPX appear competitive, noncompetitive, and uncompetitive depending on the strength of E-A-4NPX. E-A-substrate complexes form weakly with substrate 4-nitrophenyl-alpha-l-arabinofuranoside (4NPA), and inhibition patterns appear competitive. Biphasic inhibition by triethanolamine reveals minor (<0.03%) contamination of E186A preparations (including a His-Tagged form) by wild-type-like enzyme, likely originating from translational misreading. Aminoalcohols are useful in probing glycoside hydrolases; they cause artifacts when used unwarily as buffer components.

Beta-D-xylosidase from Selenomonas ruminantium: thermodynamics of enzyme-catalyzed and noncatalyzed reactions.

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. Temperature dependence for hydrolysis of 4-nitrophenyl-beta-D-xylopyranoside (4NPX), 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA), and 1,4-beta-D-xylobiose (X2) was determined on and off (k (non)) the enzyme at pH 5.3, which lies in the pH-independent region for k (cat) and k (non). Rate enhancements (k (cat)/k (non)) for 4NPX, 4NPA, and X2 are 4.3 x 10(11), 2.4 x 10(9), and 3.7 x 10(12), respectively, at 25 degrees C and increase with decreasing temperature. Relative parameters k (cat) (4NPX)/k (cat) (4NPA), k (cat) (4NPX)/k (cat) (X2), and (k (cat)/K (m))(4NPX)/(k (cat)/K (m))(X2) increase and (k (cat)/K (m))(4NPX)/(k (cat)/K (m))(4NPA), (1/K (m))(4NPX)/(1/K (m))(4NPA), and (1/K (m))(4NPX)/(1/K (m))(X2) decrease with increasing temperature.