A Strategy for Neuraminidase Inhibitors Using Mechanism‐Based Labeling Information

A potent inhibitor for Vibrio cholerae neuraminidase (VCNA) was developed by using a novel two‐step strategy, a target amino acid validation using mechanism‐based labeling information, and a potent inhibitor search using a focused library. The labeling information suggested the hidden dynamics of a loop structure of VCNA, which can be a potential target of the novel inhibitor. A focused library composed of 187 compounds was prepared from a 9‐azide derivative of 2,3‐dehydro‐N‐acetylneuraminic acid (DANA) to interrupt the function of the loop of the labeled residues. Inhibitor 3c showed potent inhibition properties and was the strongest inhibitor with FANA, a N‐trifluoroacetyl derivative of DANA. Validation studies of the inhibitor with a detergent and a Lineweaver–Burk plot suggested that the 9‐substitution group would interact hydrophobically with the target loop moiety, adding a noncompetitive inhibition property to the DANA skeleton. This information enabled us to design compound 4 having the combined structure of 3c and FANA. Compound 4 showed the most potent inhibition (K_i=73 nM , mixed inhibition) of VCNA with high selectivity among the tested viral, bacterial, and mammal neuraminidases.     

Serendipitous Discovery of a Potent Influenza Virus A Neuraminidase Inhibitor

We have previously reported a potent neuraminidase inhibitor that comprises a carbocyclic analogue of zanamivir in which the hydrophilic glycerol side chain is replaced by the hydrophobic 3‐pentyloxy group of oseltamivir. This hybrid inhibitor showed excellent inhibitory properties in the neuraminidase inhibition assay (K_i=0.46 nM ; K_{i (zanamivir)}=0.16 nM ) and in the viral replication inhibition assay in cell culture at 10^?8 M . As part of this lead optimization, we now report a novel spirolactam that shows comparable inhibitory activity in the cell culture assay to that of our lead compound at 10^?7 M . The compound was discovered serendipitously during the attempted synthesis of the isothiourea derivative of the original candidate. The X‐ray crystal structure of the spirolactam in complex with the N8 subtype neuraminidase offers insight into the mode of inhibition.     

Combining Medium Effects and Cofactor Catalysis: Metal‐Coordinated Synzymes Accelerate Phosphate Transfer by 108

The systematic exploration of the modification of polyethylene imine with guanidinium and octyl groups has led to the identification of a catalyst, CD6, which accelerates the phosphate transfer reaction of HPNP (2‐hydroxypropyl‐4‐nitrophenyl phosphate) in the presence of divalent metals such as Zn²⁺, Co²⁺, Mg²⁺ or Ni²⁺. CD6 exhibits saturation kinetics that are described by Michaelis–Menten parameters K_m ranging from 2.5–8 mM and k_cat ranging from 0.0014–0.09 s^?1. For Zn^II–CD6 this corresponds to an overall acceleration k_cat/k_uncat of 3.8×10⁵ and a catalytic proficiency (k_cat/K_m)/k_uncat of 1.5×10⁸. Catalysis by Zn^II–CD6 is specifically inhibited by inorganic phosphate, allowing turnover regulation by product inhibition. This effect stands in contrast to Zn^II‐catalysed transesterification of HPNP in water or by the synzymes Co^II–CD6 and Ni^II–CD6, with which no such interference by product is observed. These characteristics render synzyme Zn^II–CD6 an efficient enzyme model that reflects enzyme‐like properties in a wide range of features.     

Ring‐Closing Metathesis Reactions: Interpretation of Conversion–Time Data

Conversion–time data were recorded for various ring‐closing metathesis (RCM) reactions that lead to five‐ or six‐membered cyclic olefins by using different precatalysts of the Hoveyda type. Slowly activated precatalysts were found to produce more RCM product than rapidly activated complexes, but this comes at the price of slower product formation. A kinetic model for the analysis of the conversion–time data was derived, which is based on the conversion of the precatalyst (Pcat) into the active species (Acat), with the rate constant k_act, followed by two parallel reactions: 1) the catalytic reaction, which utilizes Acat to convert reactants into products, with the rate k_cat, and 2) the conversion of Acat into the inactive species (Dcat), with the rate k_dec. The calculations employ two experimental parameters: the concentration of the substrate (c(S)) at a given time and the rate of substrate conversion (?dc(S)/dt). This provides a direct measure of the concentration of Acat and enables the calculation of the pseudo‐first‐order rate constants k_act, k_cat, and k_dec and of k_S (for the RCM conversion of the respective substrate by Acat). Most of the RCM reactions studied with different precatalysts are characterized by fast k_cat rates and by the k_dec value being greater than the k_act value, which leads to quasistationarity for Acat. The active species formed during the activation step was shown to be the same, regardless of the nature of different Pcats. The decomposition of Acat occurs along two parallel pathways, a unimolecular (or pseudo‐first‐order) reaction and a bimolecular reaction involving two ruthenium complexes. Electron‐deficient precatalysts display higher rates of catalyst deactivation than their electron‐rich relatives. Slowly initiating Pcats act as a reservoir, by generating small stationary concentrations of Acat. Based on this, it can be understood why the use of different precatalysts results in different substrate conversions in olefin metathesis reactions.     

Computation of the binding affinities of catechol‐O‐methyltransferase inhibitors: Multisubstate relative free energy calculations

Alchemical free energy simulations are amongst the most accurate techniques for the computation of the free energy changes associated with noncovalent protein–ligand interactions. A procedure is presented to estimate the relative binding free energies of several ligands to the same protein target where multiple, low‐energy configurational substates might coexist, as opposed to one unique structure. The contributions of all individual substates were estimated, explicitly, with the free energy perturbation method, and combined in a rigorous fashion to compute the overall relative binding free energies and dissociation constants. It is shown that, unless the most stable bound forms are known a priori, inaccurate results may be obtained if the contributions of multiple substates are ignored. The method was applied to study the complex formed between human catechol‐O‐methyltransferase and BIA 9‐1067, a newly developed tight‐binding inhibitor that is currently under clinical evaluation for the therapy of Parkinson's disease. Our results reveal an exceptionally high‐binding affinity (K_d in subpicomolar range) and provide insightful clues on the interactions and mechanism of inhibition. The inhibitor is, itself, a slowly reacting substrate of the target enzyme and is released from the complex in the form of O‐methylated product. By comparing the experimental catalytic rate (k_cat) and the estimated dissociation rate (k_off) constants of the enzyme‐inhibitor complex, one can conclude that the observed inhibition potency (K_i) is primarily dependent on the catalytic rate constant of the inhibitor's O‐methylation, rather than the rate constant of dissociation of the complex. © 2012 Wiley Periodicals, Inc.     

Inhibition of Diamino Pelargonic Acid Aminotransferase,an Enzyme of the Biotin Biosynthetic Pathway,by Amiclenomycin: A Mechanistic Study

The mechanism of action of amiclenomycin ( 1a ), a naturally occuring inhibitor of diaminopelargonic acid aminotransferase, has been established. The enzyme catalyzes the formation of an aromatic adduct between the inhibitor and pyridoxal‐5′‐phosphate. The structure of the adduct, determined by mass spectrometry, is in agreement with the reported X‐ray crystal structure. Kinetic parameters, characteristic of k_cat inhibitors, have been observed, with a K_I value of 2 μM and a k_inact value of 0.4 min^?1. The irreversibility of the inactivation observed, in spite of the absence of covalent bond between the inhibitor and the protein, reveals the high affinity of the adduct for the active site. Two other cis‐1‐amino‐4‐substituted‐cyclohexa‐2,5‐dienes, 3a and 4a , were also found to efficiently inhibit the enzyme. The trans‐isomers were either much less potent ( 1b ) or inactive ( 3b and 4b ). The aminocyclohexadiene moiety, which is, apparently, responsible for the inhibition, could constitute an original pharmacophore for the design of new herbicides.     

Discovery of Selective Small‐Molecule Activators of a Bacterial Glycoside Hydrolase

Fragment‐based approaches are used routinely to discover enzyme inhibitors as cellular tools and potential therapeutic agents. There have been few reports, however, of the discovery of small‐molecule enzyme activators. Herein, we describe the discovery and characterization of small‐molecule activators of a glycoside hydrolase (a bacterial O‐GlcNAc hydrolase). A ligand‐observed NMR screen of a library of commercially available fragments identified an enzyme activator which yielded an approximate 90 % increase in k_cat/K_M values (k_cat=catalytic rate constant; K_M=Michaelis constant). This compound binds to the enzyme in close proximity to the catalytic center. Evolution of the initial hits led to improved compounds that behave as nonessential activators effecting both K_M and V_max values (V_max=maximum rate of reaction). The compounds appear to stabilize an active “closed” form of the enzyme. Such activators could offer an orthogonal alternative to enzyme inhibitors for perturbation of enzyme activity in vivo, and could also be used for glycoside hydrolase activation in many industrial processes.     

Helical self‐assembly of 2‐(1,4,7,10‐tetraazacyclododecan‐1‐yl)cyclohexan‐1‐ol (cycyclen)

The title macrocyclic amino alcohol compound, C₁₄H₃₀N₄O, is investigated as a solid‐state synthon for the design of a self‐assembled tubular structure. It crystallizes in a helical column constructed by stereospecific O—H...N and N—H...N interactions. The hydrogen‐bonding interactions, dependent upon macrocyclic ring helicity and molecular conformation, link R,R and S,S enantiomers in a head‐to‐tail fashion, forming a continuous hydrophilic inner core.     

Catalytic self-assembled monolayers on Au nanoparticles: the source of catalysis of a transphosphorylation reaction

The catalytic activity of a series of Au monolayer protected colloids (Au MPCs) containing different ratios of the catalytic unit triazacyclononane?Zn^II (TACN?Zn^II) and an inert triethyleneglycol (TEG) unit was measured. The catalytic self‐assembled monolayers (SAMs) are highly efficient in the transphosphorylation of 2‐hydroxy propyl 4‐nitrophenyl phosphate (HPNPP), an RNA model substrate, exhibiting maximum values for the Michaelis–Menten parameters k_cat and K_M of 6.7×10^?3 s^?1 and 3.1×10^?4 M , respectively, normalized per catalytic unit. Despite the structural simplicity of the catalytic units, this renders these nanoparticles among the most active catalysts known for this substrate. Both k_cat and K_M parameters were determined as a function of the mole fraction of catalytic unit (x ₁ ) in the SAM. Within this nanoparticle (NP) series, k_cat increases up till x ₁ ≈0.4, after which it remains constant and K_M decreases exponentially over the range studied. A theoretical analysis demonstrated that these trends are an intrinsic property of catalytic SAMs, in which catalysis originates from the cooperative effect between two neighboring catalytic units. The multivalency of the system causes an increase of the number of potential dimeric catalytic sites composed of two catalytic units as a function of the x ₁ , which causes an apparent increase in binding affinity (decrease in K_M). Simultaneously, the k_cat value is determined by the number of substrate molecules bound at saturation. For values of x ₁ > 0.4, isolated catalytic units are no longer present and all catalytic units are involved in catalysis at saturation. Importantly, the observed trends are indicative of a random distribution of the thiols in the SAM. As indicated by the theoretical analysis, and confirmed by a control experiment, in case of clustering both k_cat and K_M values remain constant over the entire range of x ₁ .     

Catalysis of benzisoxazole isomerization by molecularly imprinted polymers

A tailor-made catalytically active polymer catalyzing the benzisoxazole isomerization is described. Kinetic studies carried out in water/ethanol (3:1, v/v) at room temperature, showed a rate acceleration (k_MIP/k_control) of 7.2-fold compared to the control polymer. The imprinted polymer exhibits Michaelis-Menten kinetics with a K_m of 0.484 mM and a k_cat of 0.205 min⁻¹. Compared with the uncatalyzed reaction, a rate enhancement ((k_cat/K_m)/k_uncat) of 4 × 10⁴ fold was obtained. Substrate selectivity, accessible binding site analysis, dissociation constant determination, and inhibition study were also performed.     

Studies on the Selective Reduction of the Amide Link of Acyclic and Macrocyclic Amidoketals: Unexpected Cleavage and trans‐Acetalization with Red‐Al®

Selective reduction of the amide moiety of acyclic and macrocyclic amidoketals was studied in presence of various reagents (BH₃ · Me₂S, iBuAlH₂, Red‐Al^®, LiAlH₄). The best results were obtained with lithium aluminium hydride in the presence of triethylamine traces, whereas borane dimethyl sulfide gave rise to a partial ketal reduction of the acyclic compound and Red‐Al^® to a cleavage of the macrocyclic molecule accompanied by an unexpected trans‐acetalization.     

Rapid Determination of the Specificity Constant of Irreversible Inhibitors (kinact/KI) by Means of an Endpoint Competition Assay

Owing to their covalent target occupancy, irreversible inhibitors require low exposures and offer long duration, and their use thus represents a powerful strategy for achieving pharmacological efficacy. Importantly, the potency metric of irreversible inhibitors is k_inact/K_I not IC₅₀. A simple approach to measuring k_inact/K_I was developed that makes use of an irreversible probe for competitive assays run to completion against test compounds. In this system, the k_inact/K_I value of the test compound is equal to (k_inact/K_I)_probe×[probe]/IC₅₀. The advantages of this method include simplicity, high throughput, and application to all target classes, and it only requires an in‐depth kinetic evaluation of the probe.     

Fluoropolyethers end‐capped by polar functional groups. II. Effect of catalyst and reagents concentration,solvent nature,and temperature on reaction kinetics of α,ω‐bis(hydroxy)‐terminated fluoropolyethers with cycloalyphatic and aromatic diisocyanates

A comparative kinetic study of the dibutyltin dilaurate (DBTDL) and 1,4‐diazabicyclo[2,2,2]octane (DABCO) catalyzed reactions of α,ω‐bis(hydroxy)‐terminated fluoropolyethers (FPEs)—Z‐DOLs and Z‐DOL TXs—of various molecular weights and purity, with 4,4′‐dicyclohexylmethane diisocyanate (H₁₂MDI), isophorone diisocyanate (IPDI) and 2,4‐toluene diisocyanate (TDI) was carried out in different solvents. An analytical method was used to follow the kinetics of the reactions at four different temperatures. The rate of NCO disappearance measured by two independent methods—IR spectroscopy and chemical titration were found to be very close. Straight proportionality between rate constants k_cat and catalyst concentration was found. But in some cases for the DBTDL catalyzed reactions effect of catalyst saturation along with appearance of the limiting DBTDL concentration C_lim below which the rate of reaction was close to zero were observed. Reactivity of Z‐DOLs in the tin‐catalyzed urethane reactions was found to decrease with their storage time at RT due to the slow hydrolysis of the end  COOR groups impurities, which give the corresponding acids that act as a strong inhibitor of the DBTDL activity. These acid admixtures have no influence on the DABCO catalyzed reactions. For the DBTDL and DABCO catalyzed reactions of Z‐DOLs with IPDI the dependence of effective rate constants k_eff (where k_eff = k_cat · 0.01/[DBTDL] and catalyst concentration is taken in mol % based on IPDI) on total reagents concentration were found to be described by curves with a maximum. Critical reagents concentration, after which the relationship k_eff = f (C) changes from proportional to inverse proportional, seems do not substantially depend on the solvent nature. Hydrogenated analog poly(ethylene glycol) MW 400 (PEG‐400) differs greatly from Z‐DOLs: only steady decrease of k_eff was observed with increase of reagents concentration C from 5 up to 95 wt %. Activation energies for all the studied reactions are within the range of 10.8–16.7 kcal/mol. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2579–2602, 2000     

Studies towards a tailor-made catalyst for the Diels-Alder reaction using the technique of molecular imprinting

A tailor-made catalytically active polymer catalyzing the bimolecular Diels-Alder reaction is described. Kinetic studies carried out in acetonitrile at 82°C show a 270-fold rate acceleration (k_cat/k_uncat) for the Diels-Alder reaction between tetrachlorothiophene dioxide and maleic anhydride. The imprinted polymer induces Michaelis-Menten kinetics, with an apparent K_m of 42.5 mM and an apparent k_cat of 3.82 × 10⁻² min⁻¹, respectively. Substrate selectivity, accessible binding site analysis, dissociation constant determination, and inhibition study were also performed.     

Resveratrol and arctigenin production from polydatin and arctiin respectively by a thermostable β-glucosidase from Thermotoga maritima

Abstract

For preparation of resveratrol and arctigenin from peanut hulls and arctium lappa fruits, respectively, a recombinant β-glucosidase (TmBglA) from hyperthermophile Thermotoga maritima was purified and characterized. The hydrolytic activity was the highest at 90?°C and pH 6.2 for arctiin with K_m of 1.61?mM and k_cat of 197.4?s^?1, and 90?°C and 5.8 for polydatin with K_m of 0.38?mM and k_cat of 47.6?s^?1. The enzyme produced 215.4?mg L^?1 resveratrol and 355.7?mg L^?1 arctigenin from 400?mg L^?1 polydatin and 540?mg L^?1 arctiin after 60?min of incubation at 80?°C, with capable of hydrolyzing up to 92.1 and 94.9% of polydatin and arctiin, respectively. The enzymatic hydrolysis of peanut hulls and fructus arctii displayed a conversion yield of 3.8 and 0.33?mg resveratrol and arctigenin per gram of substrate material flour, respectively. Of the reported β-glucosidase, TmBglA exhibited the highest thermostability, k_cat, k_cat/K_m, and conversion productivity for hydrolyzing polydatin and arctiin, and has great potential applications in functional food and medicine production.     

Structure and Polymorphism of M(thd)3 (M = Al,Cr, Mn,Fe, Co,Ga, and In)

Formation, crystal structure, polymorphism, and transition between polymorphs are reported for M(thd)₃, (M = Al, Cr, Mn, Fe, Co, Ga, and In) [(thd)^– = anion of H(thd) = C₁₁H₂₀O₂ = 2, 2, 6, 6‐tetramethylheptane‐3, 5‐dione]. Fresh crystal‐structure data are provided for monoclinic polymorphs of Al(thd)₃, Ga(thd)₃, and In(thd)₃. Apart from adjustment of the M–O_k bond length, the structural characteristics of M(thd)₃ complexes remain essentially unaffected by change of M. Analysis of the M–O_k, O_k–C_k, and C_k–C_k distances support the notion that the M–O_k–C_k–C_k–C_k–O_k– ring forms a heterocyclic unit with σ and π contributions to the bonds. Tentative assessments according to the bond‐valence or bond‐order scheme suggest that the strengths of the σ bonds are approximately equal for the M–O_k, O_k–C_k, and C_k–C_k bonds, whereas the π component of the M–O_k bonds is small compared with those for the O_k–C_k, and C_k–C_k bonds. The contours of a pattern for the occurrence of M(thd)₃ polymorphs suggest that polymorphs with structures of orthorhombic or higher symmetry are favored on crystallization from the vapor phase (viz. sublimation). Monoclinic polymorphs prefer crystallization from solution at temperatures closer to ambient. Each of the M(thd)₃ complexes subject to this study exhibits three or more polymorphs (further variants are likely to emerge consequent on systematic exploration of the crystallization conditions). High‐temperature powder X‐ray diffraction shows that the monoclinic polymorphs convert irreversibly to the corresponding rotational disordered orthorhombic variant above some 100–150 °C (depending on M). The orthorhombic variant is in turn transformed into polymorphs of tetragonal and cubic symmetry before entering the molten state. These findings are discussed in light of the current conceptions of rotational disorder in molecular crystals.     

The kinetics of enhanced spin capturing polymerization: Influence of the nitrone structure

Several nitrones and one nitroso compound have been evaluated for their ability to control the molecular weight of polystyrene via the recently introduced radical polymerization method of enhanced spin capturing polymerization (ESCP). In this technique, molecular weight control is achieved (at ambient or slightly elevated temperatures) via the reaction of a growing radical chain with a nitrone forming a macronitroxide. These nitroxides subsequently react rapidly and irreversibly with propagating macroradicals forming polymer of a certain chain length, which depends on the nitrone concentration in the system. Via evaluation of the resulting number‐average molecular weight, M_n, at low conversions, the addition rate coefficient of the growing radicals onto the different nitrones is determined and activation energies are obtained. For the nitrones N‐tert‐butyl‐α‐phenylnitrone (PBN), N‐methyl‐α‐phenylnitrone (PMN), and N‐methyl‐α‐(4‐bromo‐phenyl) nitrone (pB‐PMN), addition rate coefficients, k_ad,macro, in a similar magnitude to the styrene propagation rate coefficient, k_p, are found with spin capturing constants C_SC (with C_SC = k_ad,macro/k_p) ranging from 1 to 13 depending on the nitrone and on temperature. Activation energies between 23.6 and 27.7 kJ mol⁻¹ were deduced for k_ad,macro, congruent with a decreasing C_SC with increasing temperature. Almost constant M_n over up to high monomer to polymer conversions is found when C_SC is close to unity, while increasing molecular weights can be observed when the C_SC is large. From temperatures of 100 °C onward, reversible cleavage of the alkoxyamine group can occur, superimposing a reversible activation/deactivation mechanism onto the ESCP system. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1098–1107, 2009     

Regulation of α‐Chymotrypsin Catalysis by Ferric Porphyrins and Cyclodextrins

Positively charged α‐chymotrypsin (ChT) formed a 1:1 complex with negatively charged 5,10,15,20‐tetrakis(4‐sulfonatophenyl)porphyrinato iron(III) (FeTPPS) in phosphate buffer at pH 7.4 through electrostatic interaction. In spite of the large binding constant (K=4.8×10⁵ M ^?1), FeTPPS could not completely inhibit the catalysis of ChT in the hydrolysis of the model substrate, N‐succinyl‐L ‐phenylalanine p‐nitroanilide (SPNA). The degree of inhibition (60 %) was saturated at 1.6 equivalents of FeTPPS, which indicates that covering of the active site of ChT by FeTPPS was insufficient. The enzymatic activity lowered by FeTPPS was entirely recovered for the freshly prepared sample when the porphyrin on the protein surface was detached by per‐O‐methylated β‐cyclodextrin (TMe‐β‐CD), which formed a stable 1:2 inclusion complex with FeTPPS (K₁=1.26×10⁶ M ^?1, K₂=6.3×10⁴ M ^?1). FeTPPS gradually induced irreversible denaturation of ChT, and the denatured ChT further lost its catalytic ability. No repairing effect of TMe‐β‐CD was observed with irreversibly denatured ChT. A new reversible inhibitor, 5,10,15,20‐tetrakis[4‐(3,5‐dicarboxyphenylmethoxy)phenyl]porphyrinato iron(III) (FeP8M), was then designed, and its inhibitory behavior was examined. FeP8M formed very stable 1:1 and 1:2 FeP8M/ChT complexes with ChT, the K₁ and K₂ values being 2.0×10⁸ and 1.0×10⁶ M ^?1, respectively. FeP8M effectively inhibited the ChT‐catalyzed hydrolysis of SPNA (maximum degree of inhibition=85 %), and the activity of ChT was recovered by per‐O‐methylated γ‐cyclodextrin. No irreversible denaturation of ChT occurred upon binding with FeP8M. The kinetic data support the observation that, for nonincubated samples, both inhibitors did not cause significant conformational change in ChT and inhibited the ChT activity by covering the active site of the enzyme.     

酪氨酸酶催化下以吡啰红为指示剂荧光测定青蒿素

Fluorescence decrease ratio （F0/F） was applied to determination of artemisinin （qinghaosu, QHS） based on the catalytic effect of tyrosinase using tetraethyldiaminoxanthenyl chloride （pyronine B, PB） as monitor. A catalyst used commonly in the decomposition of QHS, tyrosinase, exhibited higher binding activity than hemin, which was expressed as Michaelis-Menten parameters, km, Vmax, and kcat respectively. Interaction of QHS with tyrosinase was inhibited in the presence of deactivating agents at high temperature whereas enhanced by ethanol. Under optimal conditions, a concentration of 1.4×10^-7-8.4×10^-7 mol·L^-1of QHS could be determined on the basis of fluorescence decrease ratio of PB, with a detection limit 3tr of 2.6×10^-9 mol·L^-1. The proposed method was applied to detection of the concentration of QHS in the media of plasma and urine.     

Structure-function relationships of a catalytically efficient β-<Emphasis Type="SmallCaps">D</Emphasis>-xylosidase

β-d-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (k _cat, k _cat/K _m) for promoting hydrolysis of 1,4-β-d-xylooligosaccharides. ¹H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl-β-d-xylopyranoside (4NPX) and 1,4-β-d-xylobiose (X2). Progress curves of 4-nitrophenyl-β-d-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of k _cat and k _cat/K _m decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites −1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites bey ond−1 are blocked by protein and subsites bey ond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode k _cat and k _cat/K _m by <10³ and to asimilar extent for substrates 4NPX and 4-nitrophenyl-α-l-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With 4NPX and 4NPA, pH governs k _cat/K _m with pK _a values of 5.0 and 7.0 assigned to D14 and E186, respectively. k _cat (4NPX) has a pK _a value of 7.0 and k _cat (4NPA) is pH independent above pH 4.0, suggesting that the catalytically inactive, “dianionic” enzyme form (D14-E187-) binds 4NPX but not 4NPA. The mention of firm names or trade products does not imply that they are end orsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.