Active-site mapping of a Populus xyloglucan endo-transglycosylase with a library of xylogluco-oligosaccharides

Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16-34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites -1 and +1). Donor binding is dominated by the higher affinity of the XXXG moiety (G=Glcbeta(1-->4) and X=Xylalpha(1-->6)Glcbeta(1-->4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with k(cat)/K(m) values for the donor substrates showed that a GG-unit on negative and -XXG on positive subsites are the minimal requirements for activity. Subsites -2 and -3 (for backbone Glc residues) and +2' (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal=Galbeta(1-->4)) is the best donor substrate with a "blocked" nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.     

Kinetic analysis using low-molecular mass xyloglucan oligosaccharides defines the catalytic mechanism of a Populus xyloglucan endotransglycosylase

Plant XETs [XG (xyloglucan) endotransglycosylases] catalyse the transglycosylation from a XG donor to a XG or low-molecular-mass XG fragment as the acceptor, and are thought to be important enzymes in the formation and remodelling of the cellulose-XG three-dimensional network in the primary plant cell wall. Current methods to assay XET activity use the XG polysaccharide as the donor substrate, and present limitations for kinetic and mechanistic studies of XET action due to the polymeric and polydisperse nature of the substrate. A novel activity assay based on HPCE (high performance capillary electrophoresis), in conjunction with a defined low-molecular-mass XGO {XG oligosaccharide; (XXXGXXXG, where G=Glcbeta1,4- and X=[Xylalpha1,6]Glcbeta1,4-)} as the glycosyl donor and a heptasaccharide derivatized with ANTS [8-aminonaphthalene-1,3,6-trisulphonic acid; (XXXG-ANTS)] as the acceptor substrate was developed and validated. The recombinant enzyme PttXET16A from Populus tremula x tremuloides (hybrid aspen) was characterized using the donor/acceptor pair indicated above, for which preparative scale syntheses have been optimized. The low-molecular-mass donor underwent a single transglycosylation reaction to the acceptor substrate under initial-rate conditions, with a pH optimum at 5.0 and maximal activity between 30 and 40 degrees C. Kinetic data are best explained by a ping-pong bi-bi mechanism with substrate inhibition by both donor and acceptor. This is the first assay for XETs using a donor substrate other than polymeric XG, enabling quantitative kinetic analysis of different XGO donors for specificity, and subsite mapping studies of XET enzymes.     

Investigation of the functional relevance of the catalytically important Glu(28) in family 51 arabinosidases

The alpha-L-arabinofuranosidase (AbfD3) from Thermobacillus xylanilyticus is a family 51 glycosyl hydrolase. According to classification hierarchy, family 51 belongs to clan GH-A. While the major GH-A motifs, the catalytic acid-base and nucleophile, are conserved in AbfD3, a third catalytically important residue (Glu(28)) does not appear to be analogous to any known GH-A motif. To evaluate the importance of Glu(28), bioinformatics analyses and site-saturation mutagenesis were performed. The results indicate that Glu(28) forms part of a family 51 arabinosidase motif which might be functionally homologous to a conserved N-terminal motif found in exo-acting enzymes from families 1 and 5. Importantly, the data reveal that Glu(28) is a key determinant of substrate recognition in the -1 subsite, where it may also play an important role in water-mediated deglycosylation of the glycosyl-enzyme covalent intermediate.     

Structure elucidation of neutral, di-, tri-, and tetraglycosylceramides from High Five cells: identification of a novel (non-arthro-series) glycosphingolipid pathway

The major neutral glycosphingolipids (GSLs) of High Five insect cells have been extracted, purified, and characterized. It was anticipated that GSLs from High Five cells would follow the arthro-series pathway, known to be expressed by both insects and nematodes at least through the common tetraglycosylceramide (Glcbeta1Cer --> Manbeta4Glcbeta1Cer [MacCer] --> GlcNAcbeta3Manbeta4Glcbeta1Cer [At(3)Cer] --> GalNAcbeta4- GlcNAcbeta3Manbeta4Glcbeta1Cer [At(4)Cer]). Surprisingly, the structures of the major neutral High Five GSLs already diverge from the arthro-series pathway at the level of the triglycosylceramide. Studies by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS) showed the structure of the predominant High Five triglycosylceramide to be Galbeta3Manbeta4Glcbeta1Cer, whereas the predominant tetraglycosylceramide was characterized as GalNAcalpha4Galbeta3Manbeta4- Glcbeta1Cer. Both of these structures are novel products for any cell or organism so far studied. The GalNAcalpha4 and Galbeta3 units are found in insect GSLs, but always as the fifth and sixth residues linked to GalNAcbeta4 in the arthro-series penta- and hexaglycosylceramide structures (At(5)Cer and At(6)Cer, respectively). The structure of the High Five tetraglycosylceramide thus requires a reversal of the usual order of action of the glycosyltransferases adding the GalNAcalpha4 and Galbeta3 residues in dipteran GSL biosynthesis and implies the existence of an insect Galbeta3-T capable of using Manbeta4Glcbeta1Cer as a substrate with high efficiency. The results demonstrate the potential appearance of unexpected glycoconjugate biosynthetic products even in widely used but unexamined systems, as well as a potential for core switching based on MacCer, as observed in mammalian cells based on the common LacCer intermediate.     

Chemical synthesis of sulfated oligosaccharides with a beta-D-Gal-(1-->3)

The syntheses of two sulfated pentasaccharides: beta-D-Gal6SO3Na-(1-->3)-[beta-D-Gal-(1-->4)-alpha-L-Fuc-(1-->3)-beta-D-Glc-NAc-(1-->6)]-alpha-D-GalNAc-->OMe (1) and beta-D-Gal6SO3Na-(1-->3)-[beta-D-Gal-(1-->4)-alpha-L-Fuc-(1-->3)-beta-D-Glc-NAc6SO3Na-(1-->6)]-alpha-D-GalNAc-->OMe (2) by using Lewisx trisaccharides 12 and 16 as glycosyl donors are described. Sulfated oligosaccharides 1-2 and intermediate compounds are fully characterized by 2D 1H-1H DQF-COSY and 2D ROESY experiments.     

The mechanism of action of glycosidases.

The factors that may contribute to the rate enhancement observed with enzymatic versus non-enzymatic hydrolysis of glycosides are discussed. The nature of the active site as deduced from labelling studies with beta-glucosidases is described. A two-step mechanism involving either an enzyme stabilized glycosyl ion or a covalent glycosyl-enzyme intermediate is proposed. Experiments with a beta-glucosidase from almonds show that even with 2-deoxy glucosides with good leaving groups as aglycon which are hydrolyzed 1000 times more slowly than the corresponding glucosides, the deglucosylation step is faster than the cleavage of the glycosidic bond.     

Kinetic analyses of retaining endo-(xylo)glucanases from plant and microbial sources using new chromogenic xylogluco-oligosaccharide aryl glycosides

A library of phenyl beta-glycosides of xylogluco-oligosaccharides was synthesized via a chemoenzymatic approach to produce new, specific substrates for xyloglucanases. Tamarind xyloglucan was completely hydrolyzed to four, variably galactosylated component oligosaccharides based on Glc 4 backbones, using a Trichoderma endo-glucanase mixture. Oligosaccharide complexity could be further reduced by beta-galactosidase treament. Subsequent per- O-acetylation, alpha-bromination, phase-transfer glycosylation, and Zemplen deprotection yielded phenyl glycosides of XXXG and XLLG oligosaccharides with a broad range of aglycon p K a values. Kinetic and product analysis of the action of the archetypal plant endo-xyloglucanase, Tropaeolum majus NXG1, on these compounds indicated that formation of the glycosyl-enzyme intermediate was rate-limiting in the case of phenol leaving groups with p K a values of >7, leading exclusively to substrate hydrolysis. Conversely, substrates with aglycon p K a values of 5.4 gave rise to a significant amount of transglycosylation products, indicating a change in the relative rates of formation and breakdown of the glycosyl-enzyme intermediate for these faster substrates. Notably, comparison of the initial rates of XXXG-Ar and XLLG-Ar conversion indicated that catalysis by TmNXG1 was essentially insensitive to the presence of galactose in the negative subsites for all leaving groups. More broadly, analysis of a selection of enzymes from CAZy families GH 5, 12, and 16 indicated that the phenyl glycosides are substrates for anomeric configuration-retaining endo-xyloglucanases but are not substrates for strict xyloglucan endo-transglycosylases (XETs). The relative activities of the GH 5, 12, and 16 endo-xyloglucanases toward GGGG-CNP, XXXG-CNP, and XLLG-CNP reflected those observed using analogous high molar mass polysaccharides. These new chromogenic substrates may thus find wide application in the discovery, screening, and detailed kinetic analysis of new xyloglucan-active enzymes.     

Analysis of the dynamic properties of Bacillus circulans xylanase upon formation of a covalent glycosyl-enzyme intermediate

NMR spectroscopy was used to search for mechanistically significant differences in the local mobility of the main-chain amides of Bacillus circulans xylanase (BCX) in its native and catalytically competent covalent glycosyl-enzyme intermediate states. 15N T1, T2, and 15N[1H] NOE values were measured for approximately 120 out of 178 peptide groups in both the apo form of the protein and in BCX covalently modified at position Glu78 with a mechanism-based 2-deoxy-2-fluoro-beta-xylobioside inactivator. Employing the model-free formalism of Lipari and Szabo, the measured relaxation parameters were used to calculate a global correlation time (tau(m)) for the protein in each form (9.2 +/- 0.2 ns for apo-BCX; 9.8 +/- 0.3 ns for the modified protein), as well as individual order parameters for the main-chain NH bond vectors. Average values of the order parameters for the protein in the apo and complexed forms were S2 = 0.86 +/- 0.04 and S2 = 0.91 +/- 0.04, respectively. No correlation is observed between these order parameters and the secondary structure, solvent accessibility, or hydrogen bonding patterns of amides in either form of the protein. These results demonstrate that the backbone of BCX is well ordered in both states and that formation of the glycosyl-enzyme intermediate leads to little change, in any, in the dynamic properties of BCX on the time scales sampled by 15N-NMR relaxation measurements.     

Differences in the recognition of glucan elicitor signals between rice and soybean: beta-glucan fragments from the rice blast disease fungus Pyricularia oryzae that elicit phytoalexin biosynthesis in suspension-cultured rice cells

Partial acid/enzymatic hydrolysis of the beta-(1-->3, 1-->6)-glucan from the cell walls of the rice blast disease fungus Pyricularia oryzae (Magnaporthe grisea) released elicitor-active fragments that induced phytoalexin biosynthesis in suspension-cultured rice cells. From the digestion of the glucan by an endo-beta-(1-->3)-glucanase, one highly elicitor-active glucopentaose was purified as a reduced compound, tetraglucosyl glucitol. The structure of this tetraglucosyl glucitol as well as two other related tetraglucosyl glucitols was elucidated as follows: (1) Glcbeta(1-->3)Glcbeta(1-->3)(Glcbeta(1-->6)) Glcbeta(1-->3)Glucitol (most active fragment); (2) Glcbeta(1-->3)(Glcbeta(1-->6))Glcbeta(1-->3)Glcbeta (1-->3)Glucitol; and (3) Glcbeta(1-->6) Glcbeta(1-->3)Glcbeta(1-->3)Glcbeta(1-->3)Glucitol. However, a synthetic hexa-beta-glucoside, known as a minimal structural element for the phytoalexin elicitor for soybean cotyledon cells, did not induce phytoalexin biosynthesis in the rice cells. Conversely, the beta-glucan fragment from P. oryzae did not induce phytoalexin biosynthesis in the soybean cotyledon cells, indicating differences in the recognition of glucooligosaccharide elicitor signals in these two plants. Because rice cells have been shown to recognize chitin fragments larger than pentamers as potent elicitors, these results also indicate that the rice cells can recognize at least two types of oligosaccharides from fungal cell walls as signal molecules to initiate defense response.     

Kinetics and thermodynamics of the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase

Thymidylate synthase (TS), 5-fluorodeoxyuridylate (FdUMP), and 5,10-methylenetetrahydrofolate (CH2-H4folate) form a covalent complex in which a Cys thiol of TS is attached to the 6-position of FdUMP and the one-carbon unit of the cofactor is attached to the 5-position. The kinetics of formation of this covalent complex have been determined at several temperatures by semirapid quench methods. Together with previously reported data the results permit calculation of every rate and equilibrium constant in the interaction. Conversion of the noncovalent ternary complex to the corresponding covalent complex proceeds at a rate of 0.6 s-1 at 25 degrees C, and the dissociation constant for loss of CH2-H4folate from the noncovalent ternary complex is approximately 1 microM. Activation parameters for the formation of the covalent complex were shown to be Ea = 20 kcal/mol, delta G+ = 17.9 kcal/mol, delta H+ = 19.3 kcal/mol, and delta S+ = 0.005 kcal/(mol.deg). The equilibrium constant between the noncovalent and covalent ternary complexes is approximately 2 X 10(4), and the overall dissociation constant of CH2-H4folate from the covalent complex is approximately 10(-11) M. The conversion of the noncovalent ternary complex to the covalent adduct is about 12-fold slower than kcat in the normal enzymic reaction. However, because the dissociation constant for CH2-H4folate from the noncovalent ternary complex is about 10-fold lower than that from the TS-dUMP-CH2-H4folate Michaelis complex, the terms corresponding to kcat/Km are nearly equal. We propose that some of the intrinsic binding energy of CH2-H4folate may be used to facilitate formation of a 5-iminium ion intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further studies on the catalytic mechanism of human liver alpha-L-fucosidase

Radiolabeling of human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) with [1-3H]conduritol C trans-epoxide revealed that there are four active sites per tetrameric enzyme complex. Solvent isotope effect experiments give evidence for a proton transfer at the rate-limiting step in catalysis. Transglycosylase activity was observed using methanol as an alternative glycone acceptor to produce methyl alpha-L-fucoside, suggesting that alpha-L-fucose is formed when water is the acceptor. Initial burst kinetics experiments suggest that a glycosyl-enzyme intermediate is formed, although the magnitude of the burst is not stoichiometric with the number of active sites. These data, along with previous results, suggest a general acid-general base catalytic mechanism involving double inversion of stereochemistry at C-1 of fucose, as well as the formation of either a covalent glycosyl-enzyme intermediate or a tight ion pair between a charged active-site residue and a hypothetical fucosyl oxocarbonium ion intermediate.     

NMR spectroscopic characterization of a beta-(1,4)-glycosidase along its reaction pathway: stabilization upon formation of the glycosyl-enzyme intermediate

NMR spectroscopy was used to search for mechanistically significant differences between the thermodynamic and dynamic properties of the 34 kDa (alpha/beta)8-barrel catalytic domain of beta-(1,4)-glycosidase Cex (or CfXyn10A) in its free (apo-CexCD) and trapped glycosyl-enzyme intermediate (2FCb-CexCD) states. The main chain chemical shift perturbations due to the covalent modification of CexCD with the mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-cellobioside are limited to residues within its active site. Thus, consistent with previous crystallographic studies, formation of the glycosyl-enzyme intermediate leads to only localized structural changes. Furthermore, 15N relaxation methods demonstrated that the backbone amide and tryptophan side chains of apo-CexCD are very well ordered on both the nanosecond to picosecond and millisecond to microsecond time scales and that these dynamic features also do not change significantly upon formation of the trapped intermediate. However, covalent modification of CexCD led to the increased protection of many amides and indoles, clustered around the active site of the enzyme, against fluctuations leading to hydrogen exchange. Similarly, thermal denaturation studies demonstrated that 2FCb-CexCD has a significantly higher midpoint unfolding temperature than apo-CexCD. The covalently modified protein also exhibited markedly increased resistance to proteolytic degradation by thermolysin relative to apo-CexCD. Thus, the local and global stability of CexCD increase along its reaction pathway upon formation of the glycosyl-enzyme intermediate, while its structure and fast time scale dynamics remain relatively unperturbed. This may reflect thermodynamically favorable interactions with the relatively rigid active site of Cex necessary to bind, distort, and subsequently hydrolyze glycoside substrates.     

Stopped-flow kinetic studies of the cellulase-catalyzed conversion of cellulose to glucose

The kinetics of the cellulase-catalyzed conversion of soluble cellulose into glucose have been studied over a range of substrate concentrations and temperatures, and at pH values ranging from 4.75 to 7.0. Lineweaver-Burk plots were linear and led to V = 6.2muM/s and K(m) = 13.1 mM at pH 5.8 and 25.0 degrees C. The pK values corresponding to the free enzyme are 4.8 and 6.8 and are consistent with carboxyl and imidazole groups as the active ionizing species. These pK values were little changed in the enzyme-substrate intermediate that reacts in the ratedetermining step, suggesting that the ionizing groups are still free in this intermediate. The activation energy corresponding to V/K(m) is 80.6 kJ/mol, and that corresponding to V is 38.7 kJ/mol. The corresponding entropies of activation are 21 J K(-1) mol(-1) and -157 J K(-1) mol(-1), respectively.     

A kinetic model for production of glucose by hydrolysis of levoglucosan and cellobiosan from pyrolysis oil

Anhydro sugars, produced during wood pyrolysis, can by hydrolyzed to sugars under acidic conditions. The acid hydrolysis of two common anhydro sugars in wood pyrolysis oils, levoglucosan (1,6-anhydro-beta-D-glucopyranose) and cellobiosan (beta-D-glucopyranosyl-(1-->4)-1,6-anhydro-D-glucopyranose), was investigated. Levoglucosan hydrolysis to glucose follows a first-order reaction, with an activation energy of 114 kJ mol(-1). For cellobiosan hydrolysis, 44% of the cellobiosan is hydrolyzed initially via the beta-(1-->4) glycosidic bond to form levoglucosan and glucose. The remaining cellobiosan is hydrolyzed initially at the 1,6 anhydro bond to form cellobiose. Both reactions are first order with respect to cellobiosan, with an activation energy of 99 kJ mol(-1). The intermediate levoglucosan and cellobiose are hydrolyzed to glucose.     

Quantum mechanics/molecular mechanics modeling of substrate-assisted catalysis in family 18 chitinases: conformational changes and the role of Asp142 in catalysis in ChiB

Family 18 chitinases catalyze the hydrolysis of β-1,4-glycosidic bonds in chitin. The mechanism has been proposed to involve the formation of an oxazolinium ion intermediate via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile (instead of an enzyme residue). Here, we have modeled the first step of the chitin hydrolysis catalyzed by Serratia marcescens chitinase B for the first time using a combined quantum mechanics/molecular mechanics approach. The calculated reaction barriers based on multiple snapshots are 15.8-19.8 kcal mol(-1) [B3LYP/6-31+G(d)//AM1-CHARMM22], in good agreement with the activation free energy of 16.1 kcal mol(-1) derived from experiment. The enzyme significantly stabilizes the oxazolinium intermediate. Two stable conformations ((4)C(1)-chair and B(3,O)-boat) of the oxazolinium ion intermediate in subsite -1 were unexpectedly observed. The transition state structure has significant oxacarbenium ion-like character. The glycosyl residue in subsite -1 was found to follow a complex conformational pathway during the reaction ((1,4)B → [(4)H(5)/(4)E](++) → (4)C(1) ? B(3,O)), indicating complex conformational behavior in glycoside hydrolases that utilize a substrate-assisted catalytic mechanism. The D142N mutant is found to follow the same wild-type-like mechanism: the calculated barriers for reaction in this mutant (16.0-21.1 kcal mol(-1)) are higher than in the wild type, in agreement with the experiment. Asp142 is found to be important in transition state and intermediate stabilization.     

Syntheses of chondroitin 4- and 6-sulfate pentasaccharide derivatives having a methyl beta-D-glucopyranosiduronic acid at the reducing end

The syntheses are reported of beta-D-GlcpA-(1-->3)-beta-D-GalpNAc-(1-->4)-beta-D-GlcpA-(1- ->3)-beta-D-GalpNAc-(1-->4)-beta-D-GlcpA-(1-->OMe), O-sulfonated at C-4 or C-6 of the aminosugar moieties, which represent structural elements of chondroitin 4- and 6-sulfate proteoglycans. Starting from a synthetic disaccharide glycosyl acceptor, the stepwise or blockwise construction of the sugar backbone with appropriate synthons led to a pentasaccharide tetraol, which was used as a common intermediate. Selective 6-O-sulfonation of this tetraol, followed by saponification, gave the 6-sulfate derivative, whereas selective 6-O-benzoylation, followed by O-sulfonation and saponification, afforded the 4-sulfate derivative as their sodium salts.     

Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific beta recombinase, and identification of a folding intermediate

Solution properties of beta recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) beta recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold beta recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N2-->2I-->2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to deltaG(tot) = 17.9 kcal/mol, with deltaG(N2-->2I) = 4.2 kcal/mol for the first transition and deltaG(I-->U) = 6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of beta recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate.     

Denaturant-induced equilibrium unfolding of concanavalin A is expressed by a three-state mechanism and provides an estimate of its protein stability

The urea and guanidine hydrochloride (GdnHCl)-induced denaturation of tetrameric concanavalin A (ConA) at pH 7.2 has been studied by using intrinsic fluorescence, 8-anilino-1-naphthalenesulfonate (ANS) binding, far-UV circular dichroism (CD), and size-exclusion chromatography. The equilibrium denaturation pathway of ConA, as monitored by steady state fluorescence, exhibits a three-state mechanism involving an intermediate state, which has been characterized as a structured monomer of the protein by ANS binding, far-UV CD and gel filtration size analysis. The three-state equilibrium is analyzed in terms of two distinct and separate dissociation (native tetramer<-->structured monomer) and unfolding (structured monomer<-->unfolded monomer) reaction steps, with the apparent transition midpoints (C(m)), respectively, at 1.4 and 4.5 M in urea, and at 0.8 and 2.4 M in GdnHCl. The results show that the free energy of stabilization of structured monomer relative to the unfolded state (-DeltaG(unf, aq)), is 4.4-5.5 kcal mol(-1), and that of native tetramer relative to structured monomer (-DeltaG(dis, aq)) is 7.2-7.4 kcal mol(-1), giving an overall free energy of stabilization (-DeltaG(dis&unf, aq)) of 11.6-12.9 kcal mol(-1) (monomer mass) for the native protein. However, the free energy preference at the level of quaternary tetrameric structure is found to be far greater than that at the tertiary monomeric level, which reveals that the structural stability of ConA is maintained mostly by subunit association.     

Recruitment of both uniform and differential binding energy in enzymatic catalysis: xylanases from families 10 and 11

The contributions of enzyme-substrate hydrogen-binding interactions to catalysis by two different families of xylanases were evaluated through kinetic studies with two representative wild-type enzymes, Cellulomonas fimi xylanase (Cex) and Bacillus circulans xylanase (Bcx), on a series of monodeoxygenated and monodeoxyfluorinated p-nitrophenyl xylobioside substrates. Effects of substitution in the distal (-2 subsite) sugar on kcat/Km for Cex were moderately large (up to 2.9 kcal mol-1), with no effect seen on kcat. By contrast, substantial effects upon both kcat and kcat/Km were seen for substrates modified in the proximal (-1 subsite) sugar. Very similar results were obtained with Bcx. Kinetic analyses with a series of eight mutants of Cex in which active site residues interacting with the substrate were mutated yielded complementary insights. Again, interactions with the distal (-2) sugar were seen to contribute substantially to kcat/Km (up to 3.7 kcal mol-1), thus to the formation of the glycosyl-enzyme intermediate, but not to kcat, thus to the hydrolysis of the glycosyl-enzyme. Interactions with the proximal (-1) sugar are much more significant, contributing up to 6.7 kcal mol-1 to both kcat/Km and kcat. These results together indicate that interactions with the distal sugar maintain similar magnitudes in the transition states for glycosylation and deglycosylation as well as in the glycosyl-enzyme intermediate and can be referred to as "uniform binding interactions" in the parlance of Albery and Knowles (Albery, W. J., and Knowles, J. R. (1976) Biochemistry 15, 5631-5640). Interactions with the proximal sugar are considerably stronger at the deglycosylation transition state than in the intermediate, and fall into the category of differential binding interactions. This behavior likely has its origins in the changes in ring conformation of the proximal sugar but not of the distal sugar between the ground state and the reaction transition state. Correlation of these individual interaction energies with the hydrogen-bonding pattern seen in the glycosyl-enzyme intermediate allows for the assignment of hydrogen-bond strengths to each interaction, with good correlation between the two approaches. These findings are relevant to the discussion of remote binding effects upon enzymatic catalysis.