The base exchange reaction of NAD+ glycohydrolase: identification of novel heterocyclic alternative substrates

ADP-ribosyl cyclase and NAD⁺ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD⁺, nicotinamide adenine dinucleotide phosphate (NADP⁺) or nicotinamide mononucleotide (NMN⁺) with an alternative base. 4′-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (k_cat/K_M = 0.9-10 μM⁻¹ s⁻¹) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the k_cat/K_M values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 μM⁻¹ s⁻¹). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN⁺, k_cat/K_M = 0.05-0.4 μM⁻¹ s⁻¹. The k_cat/K_M values for the exchange reaction with NMN⁺ were generally similar (0.04-0.12 μM⁻¹ s⁻¹) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The k_cat/K_M values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38.     

Downstream reaction of cisplatin with methionine-containing peptides: pH-dependent competition between hydrolytic cleavage and macrochelation

The pH- and time-dependent reactions of the antitumor drug cisplatin, cis-[PtCl₂(NH₃)₂], with the methionine-containing peptides Ac-Met-Gly-OH, Ac-Met-Pro-OH, Ac-Met-Pro-Gly-Gly-OH and Ac-Gly-Met-Pro-Gly-Gly-OH (Gly = glycyl, Met = d-methionyl, Pro = L-prolyl) at 313 K have been investigated by high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. As a result of the strong trans influence of the methionyl S_M atom, initial Pt-S_M binding at pH > 5 is followed by a rapid formation of tridentate machrochelates for the N-acetylated peptides. The site trans to S_M is occupied by a carboxylate O atom in the case of the κ³S_M,N_M,O_G/P macrochelates of the dipeptides and by the C-terminal glycylamide N_G2 atom for the κ³S_M,O_M,N_G2 macrochelate of Ac-Met-Pro-Gly-Gly-OH. Cisplatin simultaneously mediates the rapid hydrolytic cleavage of the Met-X (X = Gly, Pro) amide bond for both dipeptides over the whole range 2.8 ? pH ? 10.0. The released amino acids X react with the resulting κ²S_M, N_M chelate of N-acetylmethionine to afford mixed κS_M:κ²N_x,O_x complexes of the type cis-[Pt(NH₃)(Ac-Met-OH-κS)(H-X-O-κ²N_x,O_x)]⁺ as final products at pH < 5 for X = Gly and pH < 8 for X = Pro. In contrast to the dipeptides, hydrolytic cleavage of the Met-Pro amide bond in Ac-Met-Pro-Gly-Gly-OH at pH > 5 is significantly inhibited by the presence of high concentrations of the macrochelate [Pt(NH₃)(Ac-Met-Pro-Gly-Gly)-κ³S_M,O_M,N_G2]⁺. Downstream hydrolysis of the Met-Gly amide bond is competitive with upstream Ac-Gly cleavage for Ac-Gly-Met-Pro-Gly-Gly-OH at pH < 4.5.     

Kinetics and mechanism of the reactions of ozone with superoxo, hydroperoxo, and hydrido complexes of rhodium(III)

Oxidation of the title complexes with ozone takes place by hydrogen atom, hydride, and electron transfer mechanisms. The reaction with (NH₃)₄(H₂O)RhH²⁺ is a two electron process, believed to involve hydride transfer with a rate constant k = (2.2 ± 0.2) × 10⁵ M⁻¹ s⁻¹ and an isotope effect k_H/k_D = 2. The oxidation of (NH₃)₄(H₂O)RhOOH²⁺ to (NH₃)₄(H₂O)RhOO²⁺ by an apparent hydrogen atom transfer is quantitative and fast, k = (6.9 ± 0.3) × 10³ M⁻¹ s⁻¹, and constitutes a useful route for the preparation of the superoxo complex. The latter is also oxidized by ozone, but more slowly, k = 480 ± 50 M⁻¹ s⁻¹.     

Direct electron transfer and bioelectrocatalysis of hemoglobin at a carbon nanotube electrode

A stable suspension of carbon nanotube (CNT) can be obtained by dispersing the CNT in the solution of the surfactant cetyltrimethylammonium bromide. CNT has promotion effects on the direct electron transfer of hemoglobin (Hb), which was immobilized onto the surface of CNT. The direct electron transfer rate of Hb was greatly enhanced after it was immobilized onto the surface of CNT. Cyclic voltammetric results showed a pair of well-defined redox peaks, which corresponded to the direct electron transfer of Hb, with the formal potential (E^0′) at about −0.343 V (vs. saturated calomel electrode) in the phosphate buffer solution (pH 6.8). The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k_s) and the value of formal potential (E^0′) were estimated. The dependence of E^0′ on solution pH indicated that the direct electron transfer reaction of Hb is a one-electron transfer coupled with a one-proton transfer reaction process. The experimental results also demonstrated that the immobilized Hb retained its bioelectrocatalytic activity to the reduction of H₂O₂. The electrocatalytic current was proportional to the concentration of H₂O₂ at least up to 20 mM.     

The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2

Peroxiredoxin 2 (Prx2) is a 2-Cys peroxiredoxin extremely abundant in the erythrocyte. The peroxidase activity was studied in a steady-state approach yielding an apparent K_M of 2.4 μM for human thioredoxin and a very low K_M for H₂O₂ (?0.7 μM). Rate constants for the reaction of peroxidatic cysteine with the peroxide substrate, H₂O₂ or peroxynitrite, were determined by competition kinetics, k₂ = 1.0 × 10⁸ and 1.4 × 10⁷ M⁻¹ s⁻¹ at 25 °C and pH 7.4, respectively. Excess of both oxidants inactivated the enzyme by overoxidation and also tyrosine nitration and dityrosine were observed with peroxynitrite treatment. Prx2 associates into decamers (5 homodimers) and we estimated a dissociation constant K_d < 10⁻²³ M⁴ which confirms the enzyme exists as a decamer in vivo. Our kinetic results indicate Prx2 is a key antioxidant enzyme for the erythrocyte and reveal red blood cells as active oxidant scrubbers in the bloodstream.     

Exploring the catalytic potential of the 3-His mononuclear nonheme Fe(II) center: discovery and characterization of an unprecedented maltol cleavage activity

Mononuclear nonheme iron enzymes (MNHEs) catalyze a range of very diverse reactions in O₂ metabolism, but they share a common principle active-site organization. To investigate a putative catalytic promiscuity of these enzymatic metal centers, we studied the reactivity of the 3-His ligated metal center of diketone cleaving enzyme (Dke1) toward non-native substrates, with a focus on alternative O₂ dependent reactions. From a screening approach, which aims at eliminating steric factors by including minimal substrate-substructures, three alternative, ‘non-β-dicarbonyl-cleavage’ reactions are identified, among them an unprecedented oxygenation of maltol. Maltol cleavage is characterized by steady state and fast kinetic measurements and shows an O₂ concentration dependent rate determining step k_cat/K_M(O₂) of 0.3 mM^− 1 s^− 1 and a strict coupling of O₂ reduction and substrate oxidation. Furthermore, the catalytic potential of the 3-His metal center for O₂ dependent catechol ring-cleavage and phenylpyruvate oxidation (PP) is demonstrated.     

Bicarbonate-catalyzed hydrogen peroxide oxidation of cysteine and related thiols

The effect of bicarbonate on the rates of the H₂O₂ oxidation of cysteine, gluthathione, and N-acetylcysteine to the corresponding disulfides was investigated. The relative oxidation rates at pH 8 for the different thiols are inversely related to the pK_a values of the thiol groups, and the reactive nucleophiles are identified as the thiolate anions or their kinetic equivalents. The second-order rate constants at 25 °C for the reaction of the thiolate anions with hydrogen peroxide are 17 ± 2 M⁻¹ s⁻¹ for all three substrates. In the presence of bicarbonate (>25 mM), the observed rate of thiolate oxidation is increased by a factor of two or more, and the catalysis is proposed to be associated with the formation of peroxymonocarbonate from the equilibrium reaction of hydrogen peroxide with bicarbonate (via CO₂). The calculated second-order rate constants for the direct reaction of the three thiolate anions with peroxymonocarbonate fall within the range of 900-2000 M⁻¹ s⁻¹. Further oxidation of disulfides by peroxymonocarbonate results in the formation of thiosulfonate and sulfonate products. These results strongly suggest that peroxymonocarbonate should be considered as a reactive oxygen species in aerobic metabolism with relevance in thiol oxidations.     

A pathway for protons in nitric oxide reductase from Paracoccus denitrificans

Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N₂O (2NO + 2e⁻ + 2H⁺ → N₂O + H₂O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O₂-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O₂, we demonstrate that protons are indeed consumed from the ‘outside’. First, multiple turnover reduction of O₂ resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O₂ shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O₂ as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O₂ show electron transfer coupled to proton uptake from outside the NOR-liposomes with a τ = 15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Ädelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.     

Structural analysis and photoluminescence properties of low dimensional lanthanide tetracyanometallates

The synthesis of a number of lanthanide tetracyanometallate (TCM) compounds have been carried out by reaction of Ln³⁺ nitrate salts and potassium tetracyanometallates in solvent systems containing dimethylsulfoxide and water. These reactions result in the isolation of three distinct structure types: (1) monoclinic [Ln(DMSO)₄(H₂O)₃M(CN)₄](M(CN)₄)_0.5·2H₂O (Ln = Eu, Tb and M = Pd, Pt), (2) orthorhombic {La(DMSO)₃(H₂O)₂(NO₃)M(CN)₄}_∞·H₂O (M = Pd, Pt), and (3) orthorhombic {Ln(DMSO)₃(H₂O)(NO₃)M(CN)₄}_∞ (Ln = Tb and M = Pd, Pt; Ln = Er, Yb and M = Pt) in the form of single crystals. Single-crystal X-ray diffraction has been used to investigate their structural features. Structure type 1 is a zero dimensional ionic compound with a M/Ln ratio of 1.5:1. It contains coordinated as well as uncoordinated [M(CN)₄]²⁻ (M = Pd, Pt) anions and features relatively long platinophilic interactions. Structure types 2 and 3 differ quite drastically from structure type 1, but they are very similar to each other. Both of the latter are one-dimensional in nature due to chains containing linkage of Ln³⁺ coordination spheres with trans-bridging [M(CN)₄]²⁻ anions. These coordination polymers both have a M/Ln ratio of 1:1, a lack of platinophilic interactions, and incorporation of a bidentate NO₃⁻ for charge balance. Photoluminescence properties for select Eu³⁺ and Tb³⁺ compounds have been investigated. They show characteristic absorption and emission for the Ln³⁺ ions, but no significant influence of the tetracyanometallate anions.     

High-valent bis(oxo)-bridged dinuclear manganese oxamates: Synthesis, crystal structures, magnetic properties, and electronic structure calculations of bis(μ-oxo)dimanganese(IV) complexes with a binucleating o-phenylenedioxamate ligand

Two novel bis(oxo)-bridged dinuclear manganese(IV) complexes with the binucleating ligand o-phenylenebis(oxamate) (opba), formulated as (Me₄N)₄[Mn₂O₂(opba)₂] (1a) and (Me₄N)₂(Ph₄P)₂[Mn₂O₂(opba)₂] · 8H₂O (1b), have been synthesized and characterized structurally and magnetically. Like the parent complex (Ph₄P)₄[Mn₂O₂(opba)₂] · 4H₂O (1c), they possess unique Mn₂(μ-O)₂ bridging cores with two additional o-phenylenediamidate bridges which lead to exceptionally short Mn-Mn distances (2.63-2.67 Å) and fairly bent Mn-O-Mn angles (93.8-95.5°). Complexes 1a-c show a moderate to strong antiferromagnetic coupling between the two high-spin Mn^IV ions through the bis(oxo)bis(o-phenylenediamidate) quadruple bridge (−J = 70-164 cm⁻¹; H = −JS₁ · S₂). Along this series, the −J values increase with the shortening of the Mn-Mn distance and/or the lessening of the Mn-O-Mn angle. Electronic structure calculations on model complexes reproduce the observed dependence of −J with the Mn-O-Mn angle at short intermetallic distances, and reveal that the magnetic coupling is dominated by the in-plane 3d_x²-y² type superexchange pathway via the oxo bridges with a small but nonnegligible contribution from direct 3d_x²-y² type Mn-Mn σ-bond.     

Synthesis, structure and magnetic properties of an oxo-bridged dinuclear iron(III) complex [(TPyA)FFeOFeF(TPyA)](BF4)2 · 0.5MeOH (TPyA = tris(2-pyridylmethyl)amine) containing the FFeOFeF linkage

The reaction of [Fe^II(H₂O)₆](BF₄)₂ with tris(2-pyridylmethyl)amine (TPyA) and triethylamine in methanol under aerobic conditions forms [(TPyA)FFe^IIIOFe^IIIF(TPyA)](BF₄)₂ · 0.5MeOH (1), in which each Fe(III) ion is coordinated to a TPyA and an F⁻ ion as well as an oxo ion (O²⁻) linking two Fe(III) ions. 1 has offset face-to-face π-π interactions between the dimers, and possesses a supramolecular network structure. The magnetic susceptibility of 1 can be fit with g = 2.0, J/k_B = − 153 K (106 cm⁻¹), and θ = − 0.3 K [H = − 2JS_a · S_b]. These indicate that very strong antiferromagnetic interactions occur via the oxo bridge within the Fe(III) dimer and weak antiferromagnetic interactions between the dimers.     

Role of Phe113 at the distal side of the heme domain of an oxygen-sensor (Ec DOS) in the characterization of the heme environment

The heme-based oxygen-sensor enzyme from Escherichia coli (Ec DOS) is a heme-regulated phosphodiesterase with activity on cyclic-di-GMP and is composed of an N-terminal heme-bound sensor domain with the PAS structure and a C-terminal functional domain. The activity of Ec DOS is substantially enhanced by the binding of O₂ to the Fe(II)-protoporphyrin IX complex [Fe(II) complex] in the sensor domain. The binding of O₂ to the Fe(II) complex changes the structure of the sensor domain, and this altered structure becomes a signal that is transduced to the functional domain to trigger catalysis. The first step in intra-molecular signal transduction is the binding of O₂ to the Fe(II) complex, and detailed elucidation of this molecular mechanism is thus worthy of exploration. The X-ray crystal structure reveals that Phe113 is located close to the O₂ molecule bound to the Fe(II) complex in the sensor domain. Here, we found that the O₂ association rate constants (>200 × 10⁻³ μM⁻¹ s⁻¹: F113L; 26 × 10⁻³ μM⁻¹ s⁻¹: F113Y) of the Fe(II) complexes of Phe113 mutants were markedly different from that (51 × 10⁻³ μM⁻¹ s⁻¹) of the wild-type enzyme, and auto-oxidation rates (0.00068 min⁻¹: F113L; 0.039 min⁻¹: F113Y) of the Phe113 mutants also differed greatly from that (0.0062 min⁻¹) of the wild-type enzyme. We thus suggest that Phe113, residing near the O₂ molecule, has a critical role in optimizing the Fe(II)-O₂ complex for effective regulation of catalysis by the oxygen-sensor enzyme. Interactions of CO and cyanide anion with the mutant proteins were also studied.     

The new fluorogenic substrates of neutrophil proteinase 3 optimized in prime site region

Previously selected by the combinatorial chemistry approach, potent fluorogenic substrate of proteinase 3 was used as the starting structure to design new substrates. The general formula of the synthesized peptides is as follows: ABZ-Tyr-Tyr-Abu-ANB-X-NH₂, where ANB (5-amino-2-nitrobenzoic acid) served as a chromophore and an acceptor of fluorescence, ABZ (aminobenzoic acid) is a donor of fluorescence in these fluorescence resonance energy transfer (FRET) peptides, and X is a proteinogenic amino acid (except Cys). The introduced modifications influenced substrate activity of the synthesized peptides. The highest value of specificity constant for proteinase 3 was obtained for the single peptide with Gln in the discussed position (k_cat/K_M = 275,000 M⁻¹ s⁻¹), which was nearly twice as active as the reference compound (lacking a substituent in the X position). In addition, more efficient energy transfer was observed, due mainly to the bathochromic effect for the introduced modification. This approach opens a new possibility to design potent and highly specific substrates of proteinase 3 and other proteinases optimized in the prime site region.     

Design of selective substrates of proteinase 3 using combinatorial chemistry methods

In this study, chemical synthesis of the selective chromogenic/fluorogenic substrates for proteinase 3 is described. The substrates’ sequence was obtained using combinatorial chemistry methods. Deconvolution of the tripeptide library against proteinase 3 with general formula ABZ-X₃-X₂-X₁-ANB-NH₂ yielded the active sequence. Selected peptide was further modified on its C terminus to investigate the impact of chromophore moiety modification on enzyme-substrate interaction. To determine specificity, activity of selected substrates was characterized against proteinase 3 and neutrophil elastase. Finally, the peptide ABZ-Tyr-Tyr-Abu-ANB-NH₂ displayed the highest value of specificity constant (k_cat/K_M = 189 × 10³ M⁻¹ s⁻¹) for proteinase 3. To the best of our knowledge, this is the first short peptide that undergoes selective proteolysis by proteinase 3 and displays no significant hydrolysis in the presence of human neutrophil elastase and cathepsin G.     

Probing hydrogen peroxide oxidation kinetics of wild-type Synechocystis catalase-peroxidase (KatG) and selected variants

Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases that exhibit peroxidase and substantial catalase activities. Nevertheless, the reaction pathway of hydrogen peroxide dismutation, including the electronic structure of the redox intermediate that actually oxidizes H₂O₂, is not clearly defined. Several mutant proteins with diminished overall catalase but wild-type-like peroxidase activity have been described in the last years. However, understanding of decrease in overall catalatic activity needs discrimination between reduction and oxidation reactions of hydrogen peroxide. Here, by using sequential-mixing stopped-flow spectroscopy, we have investigated the kinetics of the transition of KatG compound I (produced by peroxoacetic acid) to its ferric state by trapping the latter as cyanide complex. Apparent bimolecular rate constants (pH 6.5, 20 °C) for wild-type KatG and the variants Trp122Phe (lacks KatG-typical distal adduct), Asp152Ser (controls substrate access to the heme cavity) and Glu253Gln (channel entrance) are reported to be 1.2 × 10⁴ M^− 1 s^− 1, 30 M^− 1 s^− 1, 3.4 × 10³ M^− 1 s^− 1, and 8.6 × 10³ M^− 1 s^− 1, respectively. These findings are discussed with respect to steady-state kinetic data and proposed reaction mechanism(s) for KatG. Assets and drawbacks of the presented method are discussed.     

Development of a Förster resonance energy transfer assay for monitoring bacterial collagenase triple-helical peptidase activity

Due to their efficiency in the hydrolysis of the collagen triple helix, Clostridium histolyticum collagenases are used for isolation of cells from various tissues, including isolation of the human pancreatic islets. However, the instability of clostridial collagenase I (Col G) results in a degraded Col G that has weak collagenolytic activity and an adverse effect on islet isolation and viability. A Förster resonance energy transfer triple-helical peptide substrate (fTHP) has been developed for selective evaluation of bacterial collagenase activity. The fTHP [sequence: Gly-mep-Flp-(Gly-Pro-Hyp)₄-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)₄-NH₂] had a melting temperature (T_m) of 36.2 °C and was hydrolyzed efficiently by bacterial collagenase (k_cat/K_M = 25,000 s⁻¹ M⁻¹) but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The fTHP bacterial collagenase assay allows for rapid and specific assessment of enzyme activity toward triple helices and, thus, potential application for evaluating the efficiency of cell isolation by collagenases.     

The structures and magnetism of trinuclear Ni(II), Co(II) and Mn(II) complexes derived from unsymmetrical compartmental ligands

Trinuclear Ni(II), Co(II) and Mn(II) complexes have been prepared from the asymmetric compartmental proligands 2-alkyliminomethyl-4-methyl-6-{[methyl-(2-pyridin-2-yl-ethyl)-amino]-methyl}-phenol(alkyl=ethyl, n-propyl and n-butyl) and 2-[(2-methoxy-ethylimino)-methyl]-4-methyl-6-{[methyl-(2-pyridin-2-yl-ethyl)-amino]-methyl}-phenol, which provide adjacent tridentate N₂O and bidentate NO donor sets. The crystal structures of [Ni₃(L⁶)₂(OAc)₂(NCS)₂] · CH₃OH · H₂O, [Co₃(L⁴)₂(OAc)₂(NCS)₂], and [Mn₃(L⁷)₂(OAc)₂(NCS)₂] · CH₃OH · H₂O were determined and show that the metals provide an isosceles triangle with M¹-M²=M¹-M³ ≈ 3.2 Å and M²-M³ ≈ 5.0 Å. The cryomagnetic properties of the complexes have been studied and indicate a weak antiferromagnetic interaction between the adjacent, M¹M² and M¹M³ ions with little magnetic interaction between the terminal M²M³ ions.     

Synthesis, characterization and structural analysis of isostructural dinuclear molybdenum and tungsten oxo-bis-μ-sulfido-benzenedithiolene complexes

Reactions of the molybdenum and tungsten precursors [MO₂S₂]²⁻ with equimolar amounts of benzenedithiol in acetonitrile give the title compounds [M₂O₂(μ-S)₂(bdt)₂]²⁻ with M = Mo, W and bdt = benzene-dithiolate. In case a tungsten to ligand ratio of 1:2 is used the dimer forms as well but only as a minor species whereas the monomer [WO(bdt)₂]²⁻ is the main product. In both dimeric compounds the syn-isomers are formed referring to the position of the apical oxo ligands with respect to the M₂S₂ plane. For the molybdenum compound this contrasts with a published crystal structure of the anti-isomer. Both complexes give highly symmetric isomorphous crystals but still show subtle differences in their bond lengths and angles around the central metal. The X-ray crystal structures of both are analyzed in detail and compared with each other and with the isomeric molybdenum compound. Differences and similarities between tungsten and both isomers of molybdenum complexes are shown to be more influenced by the conformation than by the central metal and a reason for the formation of syn- and anti-isomers based on the respective synthetic procedures is proposed.     

Barley xyloglucan xyloglucosyl transferases bind xyloglucan-derived oligosaccharides in their acceptor-binding regions in multiple conformational states

Three barley xyloglucan endotransglycosylases (HvXETs), known as xyloglucan xyloglucosyl transferases (EC 2.4.1.207), were subjected to kinetic and computational docking studies. The k_cat·K_m⁻¹ values with the reduced [³H]-labelled XXXG, XXLG/XLXG and XLLG acceptor substrates were 0.02 × 10⁻², 0.1 × 10⁻² and 3.2 × 10⁻² s⁻¹ μM⁻¹, while the K_m constants were 10.6, 8.6 and 5.3 mM, obtained for HvXET3, HvXET4 and HvXET6, respectively. Docking of XLLG in acceptor-binding regions revealed that at least two conformational states were likely to participate in all isoforms. The assessments of kinetic and computational data indicated that the disposition of aromatic residues at the entrance to the active sites and the flexibility of proximal COOH-terminal loops could orient acceptors more or less favourably during binding, thus leading to tighter or weaker K_m constants. The data suggested that binding of acceptors in HvXETs is guided by contributions from the conserved residues in the active sites and by the of neighbouring loops.