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1.
[reaction: see text] The synthesis of the carbasugar analogue of 1,4-anhydro-beta-d-galactopyranose, a proposed intermediate in the reaction catalyzed by uridine diphosphate-alpha-d-Galp mutase, in racemic form via Diels-Alder and Barton decarboxylation chemistry is reported. This compound was found not to inhibit the mutase from Mycobacterium tuberculosis, indicating that the enzyme does not possess a 1,4-anhydro-beta-d-galactopyranose binding pocket. 相似文献
2.
The galactofuranose moiety found in many surface constituents of microorganisms is derived from UDP-D-galactopyranose (UDP-Galp) via a unique ring contraction reaction catalyzed by UDP-Galp mutase. This enzyme, which has been isolated from several bacterial sources, is a flavoprotein. To study this catalysis, the cloned Escherichia coli mutase was purified and two fluorinated analogues, UDP-[2-F]Galf (9) and UDP-[3-F]Galf (10), were chemically synthesized. These two compounds were found to be substrates for the reduced UDP-Galp mutase with the Km values determined to be 65 and 861 microM for 9 and 10, respectively, and the corresponding kcat values estimated to be 0.033 and 5.7 s(-1). Since the fluorine substituent is redox inert, a mechanism initiated by the oxidation of 2-OH or 3-OH on the galactose moiety can thus be firmly ruled out. Furthermore, both 9 and 10 are poorer substrates than UDP-Galf, and the rate reduction for 9 is especially significant. This finding may be ascribed to the inductive effect of the 2-F substituent that is immediately adjacent to the anomeric center, and is consistent with a mechanism involving formation of oxocarbenium intermediates or transition states during turnover. Interestingly, under nonreducing conditions, compounds 9 and 10 are not substrates, but instead are inhibitors for the mutase. The inactivation by 10 is time-dependent, active-site-directed, and irreversible with a K(I) of 270 microM and a k(inact) of 0.19 min(-1). Since the K(I) value is similar to Km, the observed inactivation is unlikely a result of tight binding. To our surprise, the inactivated enzyme could be regenerated in the presence of dithionite, and the reduced enzyme is resistant to inactivation by these fluorinated analogues. It is possible that reduction of the enzyme-bound FAD may induce a conformational change that facilitates the breakdown of the putative covalent enzyme-inhibitor adduct to reactivate the enzyme. It is also conceivable that the reduced flavin bears a higher electron density at N-1, which may play a role in preventing the formation of the covalent adduct or facilitating its breakdown by charge stabilization of the oxocarbenium intermediates/transition states. Clearly, this study has led to the identification of a potent inactivator (10) for this enzyme, and study of its inactivation has also shed light on the possible mechanism of this mutase. 相似文献
3.
Amorim L Marcelo F Rousseau C Nieto L Jiménez-Barbero J Marrot J Rauter AP Sollogoub M Bols M Blériot Y 《Chemistry (Weinheim an der Bergstrasse, Germany)》2011,17(26):7345-7356
The effect of a (2,5)B boat conformation on xyloside reactivity has been investigated by studying the hydrolysis and glycosylation of a series of synthetic xyloside analogues based on a 2-oxabicyclo[2.2.2]octane framework, which forces the xylose analogue to adopt a (2,5)B conformation. The locked β-xylosides were found to hydrolyze 100-1200 times faster than methyl β-D-xylopyranoside, whereas the locked α-xylosides hydrolyzed up to 2×10(4) times faster than methyl α-D-xylopyranoside. A significant rate enhancement was also observed for the glycosylation reaction. The high reactivity of these conformers can be related to the imposition of a (2,5)B conformation, which approximates a transition state (TS) boat conformation. In this way, the energy penalty required to go from the chair to the TS conformation is already paid. These results parallel and support the observation that the GH-11 xylanase family force their substrate to adopt a (2,5)B conformation to achieve highly efficient enzymatic glycosidic bond hydrolysis. 相似文献
4.
Biarnés X Ardèvol A Iglesias-Fernández J Planas A Rovira C 《Journal of the American Chemical Society》2011,133(50):20301-20309
Retaining glycoside hydrolases (GHs), key enzymes in the metabolism of polysaccharides and glycoconjugates and common biocatalysts used in chemoenzymatic oligosaccharide synthesis, operate via a double-displacement mechanism with the formation of a glycosyl-enzyme intermediate. However, the degree of oxocarbenium ion character of the reaction transition state and the precise conformational itinerary of the substrate during the reaction, pivotal in the design of efficient inhibitors, remain elusive for many GHs. By means of QM/MM metadynamics, we unravel the catalytic itinerary of 1,3-1,4-β-glucanase, one of the most active GHs, belonging to family 16. We show that, in the Michaelis complex, the enzyme environment restricts the conformational motion of the substrate to stabilize a (1,4)B/(1)S(3) conformation of the saccharide ring at the -1 subsite, confirming that this distortion preactivates the substrate for catalysis. The metadynamics simulation of the enzymatic reaction captures the complete conformational itinerary of the substrate during the glycosylation reaction ((1,4)B/(1)S(3) -(4)E/(4)H(3) - (4)C(1)) and shows that the transition state is not the point of maximum charge development at the anomeric carbon. The overall catalytic mechanism is of dissociative type, and proton transfer to the glycosidic oxygen is a late event, clarifying previous kinetic studies of this enzyme. 相似文献
5.
G. Romeo M. C. Aversa P. Giannetto M. G. Vigorita P. Ficarra 《Magnetic resonance in chemistry : MRC》1979,12(10):593-597
The sterochemistry of some 1,3-dihydro-2H-1,4-benxodiazepin-2-ones, employed as psychotherapeutic agents, is deduced by proton magnetic resonance using the paramagnetic shift reagent Eu(fod)3. the lanthanide induced shifts are computer simulated on the basis of the geometric parameters of the protons in different model structures, having intermediate conformations between a cycloheptadiene- and a cyclohepatatriene-like system. N-Desmethyldiazepam shows a conformational equilibrium between two pseudoboat forms, while the 1-alkyl substituted derivatives exist, at room temperature, in olny one boat cycloheptatriene-like conformation. 相似文献
6.
Beis K Allard ST Hegeman AD Murshudov G Philp D Naismith JH 《Journal of the American Chemical Society》2003,125(39):11872-11878
The structure of Streptococcus suis serotype type 2 dTDP-d-glucose 4,6-dehydratase (RmlB) has been determined to 1.5 A resolution with its nicotinamide coenzyme and substrate analogue dTDP-xylose bound in an abortive complex. During enzyme turnover, NAD(+) abstracts a hydride from the C4' atom of dTDP-glucose-forming NADH. After elimination of water, hydride is then transferred back to the C6' atom of dTDP-4-keto-5,6-glucosene-regenerating NAD(+). Single-crystal spectroscopic studies unambiguously show that the coenzyme has been trapped as NADH in the crystal. Electron density clearly demonstrates that in contrast to native structures of RmlB where a flat nicotinamide ring is observed, the dihydropyridine ring of the reduced cofactor in this complex is found as a boat. The si face, from which the pro-S hydride is transferred, has a concave surface. Ab initio electronic structure calculations demonstrate that the presence of an internal hydrogen bond, between the amide NH on the nicotinamide ring and one of the oxygen atoms on a phosphate group, stabilizes this distorted conformation. Additionally, calculations show that the hydride donor ability of NADH is influenced by the degree of bending in the ring and may be influenced by an active-site tyrosine residue (Tyr 161). These results demonstrate the ability of dehydratase enzymes to fine-tune the redox potential of NADH through conformational changes in the nicotinamide ring. 相似文献
7.
Dr. Annika J. E. Borg M.Sc. Oriol Esquivias Dr. Joan Coines Prof. Carme Rovira Prof. Bernd Nidetzky 《Angewandte Chemie (International ed. in English)》2023,62(4):e202211937
UDP-glucuronic acid (UDP-GlcA) 4-epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4-oxidation of the substrate by NAD+ and rotation of a decarboxylation-prone β-keto acid intermediate in the active site, enabling stereoinverting reduction of the keto group by NADH. We reveal the elusive rotational landscape of the 4-keto intermediate. Distortion of the sugar ring into boat conformations induces torsional mobility in the enzyme's binding pocket. The rotational endpoints show that the 4-keto sugar has an undistorted 4C1 chair conformation. The equatorially placed carboxylate group disfavors decarboxylation of the 4-keto sugar. Epimerase variants lead to decarboxylation upon removal of the binding interactions with the carboxylate group in the opposite rotational isomer of the substrate. Substitutions R185A/D convert the epimerase into UDP-xylose synthases that decarboxylate UDP-GlcA in stereospecific, configuration-retaining reactions. 相似文献
8.
The crystal structure of a novel non-peptidic HIV-1 protease inhibitor derived by simple solid-state dimerization of 4-aryl-1,4-dihydropyridines, reveals a strained central cage and the conformation of its phenyl, benzyl, and hydroxymethylene substituents. The polycyclic cage includes two nearly flat cyclobutane rings and four fused piperidine rings in boat conformations. The cage geometry reveals two unexpected features, namely marked distortions of the valence angles in every second piperidine and a shortening of one of the cyclobutane bonds. The molecule displays exact centrosymmetry, but the central cage and the hydroxymethylene substituents also approximate the C2-symmetry of the target enzyme. The two independent hydroxyl groups are involved in intermolecular hydrogen bonding, one as a donor, the other as an acceptor. The disposition of the hydroxyl groups in the molecular framework is compatible with the dual role of the inhibitor in the active-site cavity of HIV-1 protease, whereby one OH group is hydrogen-bonded to the catalytic aspartates, whereas another one provides an interface to the locked flaps of the enzyme. 相似文献
9.
The binding of xylotetraose in different conformations to the active site of endo-1,4-beta-xylanase II (XynII) from Trichoderma reesei was studied using molecular dynamics (MD) simulations and free energy analyses employing the MM-PBSA (Molecular Mechanics-Poisson-Boltzmann Surface Area) method. MD simulations of 1 ns were done for the substrate xylotetraose having the reactive sugar, which is bound in the -1 subsite of XynII in the 4C1 (chair) and 2So (skew boat) ground state conformations, and for the transition state of the XynII catalysed hydrolysis of the beta-glycosidic linkage. According to the simulations and free energy analysis, XynII binds the substrate with the -1 sugar in the 2So conformation 59.8 kJ mol(-1) tighter than the substrate with the sugar in the 4C1 conformation. The reactive 2So conformation resembles closely the reaction transition state and has the breaking glycosidic bond in a pseudo-axial orientation ready for facile bond cleavage. The transition state was calculated to be bound 77.1 kJ mol(-1) tighter than the 4C1 ground state conformation. The molecular mechanical interaction energy between the enzyme and the reactive pyranoside unit at the -1 subsite was 75.7 kJ mol(-1) more favorable for the binding of the 2So conformation than the 2C1 conformation, explaining the clearly tighter binding of the reactive structure The results of this study indicate that in the Michaelis complex XynII, a member of the family 11 xylanase, the substrate is bound in a skew boat conformation and in the catalytic reaction, the -1 sugar proceeds from the 4C1 conformation through 2So to the transition state with the -1 sugar in the 2,5B conformation. 相似文献
10.
Experimental and structural evidence that herpes 1 kinase and cellular DNA polymerase(s) discriminate on the basis of sugar pucker 总被引:3,自引:0,他引:3
Marquez VE Ben-Kasus T Barchi JJ Green KM Nicklaus MC Agbaria R 《Journal of the American Chemical Society》2004,126(2):543-549
Two isomers of methanocarba (MC) thymidine (T), one an effective antiherpes agent with the pseudosugar moiety locked in the North (N) hemisphere of the pseudorotational cycle (1a, N-MCT) and the other an inactive isomer locked in the antipodean South (S) conformation (1b, S-MCT) were used to determine whether kinases and polymerases discriminate between their substrates on the basis of sugar conformation. A combined solid-state and solution conformational analysis of both compounds, coupled with the direct measurement of mono-, di-, and triphosphate levels in control cells, cells infected with the Herpes simplex virus, or cells transfected with the corresponding viral kinase gene (HSV-tk), suggests that kinases prefer substrates that adopt the S sugar conformation. On the other hand, the cellular DNA polymerase(s) of a murine tumor cell line transfected with HSV-tk incorporated almost exclusively the triphosphate of the locked N conformer (N-MCTTP), notwithstanding the presence of higher triphosphate levels of the S-conformer (S-MCTTP). 相似文献
11.
Audrey Caravano Christophe Ansiaux José Kovensky Stéphane P. Vincent 《Tetrahedron》2007,63(9):2070-2077
A new methodology for the preparation of carbohydrates locked in a 1,4B boat conformation is presented. The constrained molecules thus obtained are functionalized at the anomeric position by iodo-, fluoro-, hydroxyl- or/and phosphono-methylene moieties. A remarkable diastereoselective opening of these quaternary acetals was also observed under non-acidic conditions. 相似文献
12.
Kwiecien RA Khavrutskii IV Musaev DG Morokuma K Banerjee R Paneth P 《Journal of the American Chemical Society》2006,128(4):1287-1292
ONIOM calculations have provided novel insights into the mechanism of homolytic Co-C5' bond cleavage in the 5'-deoxyadenosylcobalamin cofactor catalyzed by methylmalonyl-CoA mutase. We have shown that it is a stepwise process in which conformational changes in the 5'-deoxyadenosine moiety precede the actual homolysis step. In the transition state structure for homolysis, the Co-C5' bond elongates by approximately 0.5 Angstroms from the value found in the substrate-bound reactant complex. The overall barrier to homolysis is approximately 10 kcal/mol, and the radical products are approximately 2.5 kcal/mol less stable than the initial ternary complex of enzyme, substrate, and cofactor. The movement of the deoxyadenosine moiety during the homolysis step positions the resulting 5'-deoxyadenosyl radical for the subsequent hydrogen atom transfer from the substrate, methylmalonyl-CoA. 相似文献
13.
Rahime imek Anthony Linden Cihat afak 《Acta Crystallographica. Section C, Structural Chemistry》2000,56(3):351-353
The title compound, C19H18BrNO3, has potential calcium modulatory properties. The 1,4‐dihydropyridine ring has a very shallow boat conformation and is one of the most planar examples of this moiety. The 2‐bromophenyl substituent is in the axial synperiplanar orientation. The quinoline ring has a half‐chair conformation, with the unusual arrangement of the out‐of‐plane atom being on the opposite side of the ring plane to the bromophenyl substituent. The molecules are linked into chains by intermolecular hydrogen bonds. 相似文献
14.
Kawatkar SP Kuntz DA Woods RJ Rose DR Boons GJ 《Journal of the American Chemical Society》2006,128(25):8310-8319
The X-ray crystal structures of mannose trimming enzyme drosophila Golgi alpha-mannosidase II (dGMII) complexed with the inhibitors mannostatin A (1) and an N-benzyl analogue (2) have been determined. Molecular dynamics simulations and NMR studies have shown that the five-membered ring of mannostatin A is rather flexible occupying pseudorotational itineraries between 2T3 and 5E, and 2T3 and 4E. In the bound state, mannostatin A adopts a 2T1 twist envelope conformation, which is not significantly populated in solution. Possible conformations of the mannosyl oxacarbenium ion and an enzyme-linked intermediate have been compared to the conformation of mannostatin A in the cocrystal structure with dGMII. It has been found that mannostatin A best mimics the covalent linked mannosyl intermediate, which adopts a 1S5 skew boat conformation. The thiomethyl group, which is critical for high affinity, superimposes with the C-6 hydroxyl of the covalent linked intermediate. This functionality is able to make a number of additional polar and nonpolar interactions increasing the affinity for dGMII. Furthermore, the X-ray structures show that the environment surrounding the thiomethyl group of 1 is remarkably similar to the arrangements around the methionine residues in the protein. Collectively, our studies contradict the long held view that potent inhibitors of glycosidases must mimic an oxacarbenium ion like transition state. 相似文献
15.
The effect of benzannelation on the equilibrium conformation and flexibility of the dihydrocycle in cyclohexa-1,4-dienone,para-quinone, and their imino and methylene analogs was studied by the semiempirical quantum-chemical AM1 method. The equilibrium
conformations of the carbonyl derivatives are planar. In the imino- and methylene-substituted analogs, the dihydrocycle adopts
a boat conformation due to repulsions between substituted analogs, the dihydrocycle adopts a boat conformation due to repulsions
between the hydrogen atoms at the exocyclic double bond and in theperi positions of the benzene rings. Annelation of cyclohexa-2,5-dien-1-one andpara-quinone with benzene rings at the C=C double bonds causes an increase in the conformational flexibility of the partially
hydrogenated ring owing to an increase in the bending strain in the first compound and a decrease in the conjugation between
the carbonyl groups and the remaining part of the molecule in the second compound.
Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 388–390, March, 1998. 相似文献
16.
The proton NMR spectra of several 1,4-thiazinan-3-ones were analyzed using LAOCOON. The geminal and vicinal coupling constants indicate that the ring conformation of this heterocycle is a half-chair similar to δ-valerolactam. Molecular mechanics calculations show that the energy of the boat is only slightly higher than that of the half-chair. The conformational equilibria of methyl-substituted compounds were calculated by the coupling constant method. These energies are compared to those obtained by molecular mechanics. 相似文献
17.
O. V. Shishkin S. M. Desenko V. D. Orlov S. V. Lindeman Yu. T. Struchkov 《Russian Chemical Bulletin》1994,43(8):1320-1323
The equilibrium geometry of 1,4-dihydropyrimidine, 4,7-dihydro-1,2,4-triazolo[1,5-a]pyrimidine, and their alkyl (Me, Et, Pri, But) and phenyl derivatives has been calculated by molecular mechanics method. The equilibrium conformation of unsubstituted molecules is planar, but it is easily transformed to the boat conformation with a small change in the conformational energy. The effect of substituents on the geometry and conformational mobility of the dihydropyrimidine ring has been studied.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1394–1397, August, 1994. 相似文献
18.
The effect ofα-cyclodextrin (α-CD) inclusion complexation on the conformational equilibrium of cyclo- hexane wasstudied with thesemiempirical PM3 molecularorbital calculations. The calculation results indicated that the chair form of cyclohexane is 18.5 kJ·mol-1lower than that of boat one in energy, however, theα-cyclodextrin inclusion complex of boat cycl ohexane is 4.4 kJ·mol-1more stable than the complex of chair form. It demon-strated that the conformational equilibrium of cyclohexane was influenced by theα-CD inclusion complexation. Hence, caution should be given when extrapolating the conformational behaviors of the guest compounds in the supramolecular systems totheir free forms, since the interactionsbetween the host and guest significantly affect the conformation of the guest compounds. 相似文献
19.
Michael J. Cook Ghoabad Ghaem-Maghami Festus Kaberia Knut Bergesen 《Magnetic resonance in chemistry : MRC》1983,21(5):339-343
1H NMR Parameters are reported for five 1,4-dithiepan-6-ones. 1,4-Dithiepan-6-one 1-oxide exists in solution as an equilibrium involving two different twist-chair conformations, which contrasts with its conformational behaviour in the solid state. Twist-chair conformers are also adopted by other members of the series, the favoured form varying with ring substitution. The S?O bond in 1,4-dithiepan-6-one 1-oxide and in its 5,5-dimethyl analogue exhibit a preference for the pseudoaxial site. 相似文献
20.
Wendt S McCombie G Daniel J Kienhöfer A Hilvert D Zenobi R 《Journal of the American Society for Mass Spectrometry》2003,14(12):1470-1476
Electrospray time-of-flight mass spectrometry was used to quantitatively determine the dissociation constant of chorismate mutase and a transition state analogue inhibitor. This system presents a fairly complex stoichiometry because the native protein is a homotrimer with three equal and independent substrate binding sites. We can detect the chorismate mutase trimer as well as chorismate mutase-inhibitor complexes by choosing appropriate conditions in the ESI source. To verify that the protein-inhibitor complexes are specific, titration experiments with different enzyme variants and different inhibitors were performed. A plot of the number of bound inhibitors versus added inhibitor concentration revealed saturation behavior with 3:1 (inhibitor:functional trimer) stoichiometry for the TSA. The soft ESI conditions, the relatively high protein mass of 43.5 kDa, and the low charge state (high m/z) result in broad peaks, a typical problem in analyzing noncovalent protein complexes. Due to the low molecular weight of the TSA (226 Da) the peaks of the free protein and the protein with one, two or three inhibitors bound cannot be clearly resolved. For data analysis, relative peak areas of the deconvoluted spectra of chorismate mutase-inhibitor complexes were obtained by fitting appropriate peak shapes to the signals corresponding to the free enzyme and its complexes with one, two, or three inhibitor molecules. From the relative peak areas we were able to calculate a dissociation constant that agreed well with known solution-phase data. This method may be generally useful for interpreting mass spectra of noncovalent complexes that exhibit broad peaks in the high m/z range. 相似文献