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Structural insights into abasic site for Fpg specific binding and catalysis: comparative high-resolution crystallographic studies of Fpg bound to various models of abasic site analogues-containing DNA

de Jésus, Karine Pereira; Serre, Laurence; Zelwer, Charles; Castaing, Bertrand
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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Fpg is a DNA glycosylase that recognizes and excises the mutagenic 8-oxoguanine (8-oxoG) and the potentially lethal formamidopyrimidic residues (Fapy). Fpg is also associated with an AP lyase activity which successively cleaves the abasic (AP) site at the 3′ and 5′ sides by βδ-elimination. Here, we present the high-resolution crystal structures of the wild-type and the P1G defective mutant of Fpg from Lactococcus lactis bound to 14mer DNA duplexes containing either a tetrahydrofuran (THF) or 1,3-propanediol (Pr) AP site analogues. Structures show that THF is less extrahelical than Pr and its backbone C5′–C4′–C3′ diverges significantly from those of Pr, rAP, 8-oxodG and FapydG. Clearly, the heterocyclic oxygen of THF is pushed back by the carboxylate of the strictly conserved E2 residue. We can propose that the ring-opened form of the damaged deoxyribose is the structure active form of the sugar for Fpg catalysis process. Both structural and functional data suggest that the first step of catalysis mediated by Fpg involves the expulsion of the O4′ leaving group facilitated by general acid catalysis (involving E2), rather than the immediate cleavage of the N-glycosic bond of the damaged nucleoside.

Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

Kraut, Daniel A; Sigala, Paul A; Pybus, Brandon; Liu, Corey W; Ringe, Dagmar; Petsko, Gregory A; Herschlag, Daniel
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing p K a models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50–0.76 ppm/p K a unit, suggesting a bond shortening of ˜0.02 Å/p K a unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (ΔΔG = −0.2 kcal/mol/p K a unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (ΔΔH = −2.0 kcal/mol/p K a unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of ˜300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution...

Efficient coupling of catalysis and dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex

Kale, Sachin; Ulas, Gözde; Song, Jaeyoung; Brudvig, Gary W.; Furey, William; Jordan, Frank
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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Protein motions are ubiquitous and are intrinsically coupled to catalysis. Their specific roles, however, remain largely elusive. Dynamic loops at the active center of the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex are essential for several catalytic functions starting from a predecarboxylation event and culminating in transfer of the acetyl moiety to the E2 component. Monitoring the kinetics of E1 and its loop variants at various solution viscosities, we show that the rate of a chemical step is modulated by loop dynamics. A cysteine-free E1 construct was site-specifically labeled on the inner loop (residues 401–413), and the EPR nitroxide label revealed ligand-induced conformational dynamics of the loop and a slow “open ↔ close” conformational equilibrium in the unliganded state. An 19F NMR label placed at the same residue revealed motion on the millisecond-second time scale and suggested a quantitative correlation of E1 catalysis and loop dynamics for the 200,000-Da protein. Thermodynamic studies revealed that these motions may promote covalent addition of substrate to the enzyme-bound thiamin diphosphate by reducing the free energy of activation. Furthermore, the global dynamics of E1 presumably regulate and streamline the catalytic steps of the overall complex by inducing an entirely entropic (nonmechanical) negative cooperativity with respect to substrate binding at higher temperatures. Our results are consistent with...

Correlated conformational fluctuations during enzymatic catalysis: Implications for catalytic rate enhancement

Alper, Kutay O.; Singla, Manish; Stone, Jennifer L.; Bagdassarian, Carey K.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /07/2001 Português
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Correlated enzymatic conformational fluctuations are shown to contribute to the rate of enhancement achieved during catalysis. Cytidine deaminase serves as a model system. Crystallographic temperature factor data for this enzyme complexed with substrate analog, transition-state analog, and product are available, thereby establishing a measure of atomic scale conformational fluctuations along the (approximate) reaction coordinate. First, a neural network-based algorithm is used to visualize the decreased conformational fluctuations at the transition state. Second, a dynamic diffusion equation along the reaction coordinate is solved and shows that the flux velocity through the associated enzymatic conformation space is greatest at the transition state. These results suggest (1) that there are both dynamic and energetic restrictions to conformational fluctuations at the transition state, (2) that enzymatic catalysis occurs on a fluctuating potential energy surface, and (3) a form for the potential energy. The Michaelis-Menten equations are modified to describe catalysis on this fluctuating potential energy profile, leading to enhanced catalytic rates when fluctuations along the reaction coordinate are appropriately correlated. This represents a dynamic tuning of the enzyme for maximally effective transformation of the ES complex into EP.

CATALYSIS BY THE SECOND CLASS OF tRNA(m1G37) METHYL TRANSFERASE REQUIRES A CONSERVED PROLINE

Christian, Thomas; Evilia, Caryn; Hou, Ya-Ming
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 20/06/2006 Português
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The enzyme tRNA(m1G37) methyl transferase catalyzes the transfer of a methyl group from S-adenosyl methionine (AdoMet) to the N1 position of G37, which is 3′ to the anticodon sequence and whose modification is important for maintaining the reading frame fidelity. While the enzyme in bacteria is highly conserved and is encoded by the trmD gene, recent studies show that the counterpart of this enzyme in archaea and eukarya, encoded by the trm5 gene, is unrelated to trmD both in sequence and in structure. To further test this prediction, we seek to identify residues in the second class of tRNA(m1G37) methyl transferase that are required for catalysis. Such residues should provide mechanistic insights into the distinct structural origins of the two classes. Using the Trm5 enzyme of the archaeon Methanocaldococcus jannaschii (previously MJ0883) as an example, we have created mutants to test many conserved residues for their catalytic potential and substrate-binding capabilities with respect to both AdoMet and tRNA. We identified that the proline at position 267 (P267) is a critical residue for catalysis, because substitution of this residue severely decreases kcat of the methylation reaction in steady-state kinetic analysis, and kchem in single turnover kinetic analysis. However...

Hydrogen Bonding Catalysis Operates by Charge Stabilization in Highly Polar Diels–Alder Reactions

Gordillo, Ruth; Dudding, Travis; Anderson, Christopher D.; Houk, K. N.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/02/2007 Português
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The alcohol-catalyzed Diels-Alder reactions of acrolein and benzaldehyde with Rawal’s diene were evaluated with density functional theory (B3LYP/6-31G(d)). Several potential modes of catalysis with two methanol molecules were used to model catalysis by TADDOLs. In agreement with crystallographic data, cooperative catalysis with TADDOLs is predicted to be favorable.

Role of Mg2+ in Hammerhead Ribozyme Catalysis from Molecular Simulation

Lee, Tai-Sung; López, Carlos Silva; Giambaºu, George M.; Martick, Monika; Scott, William G.; York, Darrin M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Molecular dynamics simulations have been performed to investigate the role of Mg2+ in the full-length hammerhead ribozyme cleavage reaction. In particular, the aim of this work is to characterize the binding mode and conformational events that give rise to catalytically active conformations and stabilization of the transition state. Toward this end, a series of eight 12 ns molecular dynamics simulations have been performed with different divalent metal binding occupations for the reactant, early and late transition state using recently developed force field parameters for metal ions and reactive intermediates in RNA catalysis. In addition, hybrid QM/MM calculations of the early and late transition state were performed to study the proton-transfer step in general acid catalysis that is facilitated by the catalytic Mg2+ ion. The simulations suggest that Mg2+ is profoundly involved in the hammerhead ribozyme mechanism both at structural and catalytic levels. Binding of Mg2+ in the active site plays a key structural role in the stabilization of stem I and II and to facilitate formation of near attack conformations and interactions between the nucleophile and G12, the implicated general base catalyst. In the transition state, Mg2+ binds in a bridging position where it stabilizes the accumulated charge of the leaving group while interacting with the 2′OH of G8...

Probing the Role of Parasite-specific, Distant, Structural Regions on Communication and Catalysis in the Bifunctional Thymidylate Synthase- Dihydrofolate Reductase from Plasmodium falciparum

Dasgupta, Tina; Anderson, Karen S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Plasmodium falciparum thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in nucleotide biosynthesis, and a validated molecular drug target in malaria. Because P. falciparum TS and DHFR are highly homologous to their human counterparts, existing active-site antifolate drugs can have dose-limiting toxicities. In humans, TS and DHFR are two separate proteins. In P. falciparum, however, TS-DHFR is bifunctional, with both TS and DHFR active sites on a single polypeptide chain of the enzyme. Consequently, P. falciparum TS-DHFR contains unique distant or ‘non-active’ regions which might modulate catalysis: 1) an N-terminal tail; and 2) a ‘linker’ region tethering DHFR to TS, and encoding a ‘crossover helix’ that forms critical electrostatic interactions with the DHFR active site. The role of these non-active sites in the bifunctional P. falciparum TS-DHFR is unknown. We report the first in-depth, pre-steady state, kinetic characterization of the full-length, WT P. falciparum TS-DHFR enzyme, and probe the role of distant, non-active regions through mutational analysis. We show that the overall rate-limiting step in the WT P. falciparum TS-DHFR enzyme is TS catalysis. We further show that if TS is in an ‘activated’ (liganded) conformation...

Crystal Structure of Biotin Carboxylase in Complex with Substrates and Implications for Its Catalytic Mechanism*

Chou, Chi-Yuan; Yu, Linda P. C.; Tong, Liang
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 24/04/2009 Português
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Biotin-dependent carboxylases are widely distributed in nature and have important functions in many cellular processes. These enzymes share a conserved biotin carboxylase (BC) component, which catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the donor. Despite the availability of a large amount of biochemical and structural information on BC, the molecular basis for its catalysis is currently still poorly understood. We report here the crystal structure at 2.0 Å resolution of wild-type Escherichia coli BC in complex with its substrates biotin, bicarbonate, and Mg-ADP. The structure suggests that Glu296 is the general base that extracts the proton from bicarbonate, and Arg338 is the residue that stabilizes the enolate biotin intermediate in the carboxylation reaction. The B domain of BC is positioned closer to the active site, leading to a 2-Å shift in the bound position of the adenine nucleotide and bringing it near the bicarbonate for catalysis. One of the oxygen atoms of bicarbonate is located in the correct position to initiate the nucleophilic attack on ATP to form the carboxyphosphate intermediate. This oxygen is also located close to the N1′ atom of biotin, providing strong evidence that the phosphate group...

Diversity of Chemical Mechanisms in Thioredoxin Catalysis Revealed by Single-Molecule Force Spectroscopy

Perez-Jimenez, Raul; Li, Jingyuan; Kosuri, Pallav; Sanchez-Romero, Inmaculada; Wiita, Arun P.; Rodriguez-Larrea, David; Chueca, Ana; Holmgren, Arne; Miranda-Vizuete, Antonio; Becker, Katja; Cho, Seung-Hyun; Beckwith, Jon; Gelhaye, Eric; Jacquot, Jean P.;
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Thioredoxins are oxido-reductase enzymes present in all organisms, catalyzing the reduction of disulfide bonds in proteins. By applying a calibrated force to a substrate disulfide, the chemical mechanisms of Trx catalysis can be examined in detail at the single molecule level. Here we use single molecule force-clamp spectroscopy to explore the chemical evolution of Trx catalysis by probing the chemistry of eight different thioredoxin enzymes. While all Trxs show a characteristic Michaelis-Menten mechanism detected when the disulfide bond is stretched at low forces, two different chemical behaviors distinguish bacterial from eukaryotic-origin Trxs at high forces. Eukaryotic-origin Trxs reduce disulfide bonds through a single-electron transfer reaction (SET) whereas bacterial-origin Trxs exhibit both nucleophilic substitution (SN2) and SET reactions. A computational analysis of Trx structures identifies the evolution of the binding groove as an important factor controlling the chemistry of Trx catalysis.

Allosteric control of catalysis by the F loop of RNA polymerase

Miropolskaya, Nataliya; Artsimovitch, Irina; Klimašauskas, Saulius; Nikiforov, Vadim; Kulbachinskiy, Andrey
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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Bacterial RNA polymerases (RNAPs) undergo coordinated conformational changes during catalysis. In particular, concerted folding of the trigger loop and rearrangements of the bridge helix at the RNAP active center have been implicated in nucleotide addition and RNAP translocation. At moderate temperatures, the rate of catalysis by RNAP from thermophilic Thermus aquaticus is dramatically reduced compared with its closest mesophilic relative, Deinococcus radiodurans. Here, we show that a part of this difference is conferred by a third element, the F loop, which is adjacent to the N terminus of the bridge helix and directly contacts the folded trigger loop. Substitutions of amino acid residues in the F loop and in an adjacent segment of the bridge helix in T. aquaticus RNAP for their D. radiodurans counterparts significantly increased the rate of catalysis (up to 40-fold at 20 °C). A deletion in the F loop dramatically impaired the rate of nucleotide addition and pyrophosphorolysis, but it had only a moderate effect on intrinsic RNA cleavage. Streptolydigin, an antibiotic that blocks folding of the trigger loop, did not inhibit nucleotide addition by the mutant enzyme. The resistance to streptolydigin likely results from the loss of its functional target...

Conformational Ensemble Modulates Cooperativity in the Rate-determining Catalytic Step in the E1 Component of the Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex*

Kale, Sachin; Jordan, Frank
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Cooperativity is extensively used by enzymes, particularly those acting at key metabolic branch points, to “fine tune” catalysis. Thus, cooperativity and enzyme catalysis are intimately linked, yet their linkage is poorly understood. Here we show that negative cooperativity in the rate-determining step in the E1 component of the Escherichia coli pyruvate dehydrogenase multienzyme complex is an outcome of redistribution of a “rate-promoting” conformational pre-equilibrium. An array of biophysical and biochemical studies indicates that non-catalytic but conserved residues directly regulate the redistribution. Furthermore, factors such as ligands and temperature, individually or in concert, also strongly influence the redistribution. As a consequence, these factors also exert their influence on catalysis by profoundly influencing the pre-equilibrium facilitated dynamics of communication between multienzyme components. Our observations suggest a mode of cooperativity in the E1 component that is consistent with the dynamical hypothesis shown to satisfactorily explain cooperativity in many well studied enzymes. The results point to the likely existence of multiple modes of communication between subunits when the entire class of thiamin diphosphate-dependent enzymes is considered.

Simultaneous Fluorescent Monitoring of Proteasomal Subunit Catalysis

Wakata, Aya; Lee, Hsien-Ming; Rommel, Philipp; Toutchkine, Alexei; Schmidt, Marion; Lawrence, David S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 10/02/2010 Português
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The proteasome, a multicatalytic protease, displays distinct chymotrypsin-like, caspase-like, and trypsin-like activities at three different subunits of the multimeric complex. Fluorescent substrates for each of these active sites have been described. However, since the fluorescent properties of these substrates are very similar, it is not possible to simultaneously monitor catalysis of two or more activities. We have developed a long wavelength (λex = 600 nm, λem = 700 nm) fluorescent substrate for the chymotrypsin-like active site via a combinatorial library strategy. This peptide-based substrate is a highly selective proteasomal chymotrypsin-like sensor, as assessed by a series of proteasomal active site mutants in yeast cell lysates. A corresponding caged analog of the sensor has been prepared, which is resistant to proteolysis until activated by 349 nm light. The latter affords the opportunity to assess proteasomal activity with a high degree of temporal control. The distinct photophysical properties of the sensor allow the chymotrypsin-like activity to be simultaneously monitored during caspase-like or trypsin-like catalysis. We have found that chymotrypsin-like activity is enhanced in the presence of the trypsin-like substrate...

Mg2+-Assisted Catalysis by B. stearothermophilus TrpRS is Promoted by Allosteric Effects

Weinreb, Violetta; Li, Li; Campbell, Cassandra L.; Kaguni, Laurie S.; Carter, Charles W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/07/2009 Português
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Mn2+-assisted catalysis by B. stearothermophilus TrpRS parallels that in polymerases and reduces specificity in amino acid activation. As predicted by non-equilibrium molecular dynamics simulations, multi-variant thermodynamic cycles with [ATP]-dependent Michaelis-Menten kinetics and Mn2+ for Mg2+ substitution demonstrate energetic coupling of ATP affinities to the metal; to lysines K111 and K192, which interact via the PPi leaving group; and to K195, which couples differently to the metal via the α–phosphate. However, net coupling to the metal opposes catalysis in both ground (Km) and transition (kcat) states. The 105-fold rate acceleration by Mg2+-protein interactions therefore requires additional favorable protein-metal couplings. Examples include longer-range, i.e., allosteric, interactions previously illustrated by the remote F37I mutation, which both reduces kcat and enhances catalytic assist by Mn2+, relative to that by Mg2+. These data support a model linking metal-assisted phosphoryl transfer catalysis to domain movement, and hence to free-energy transduction in a broad range of enzymes.

Large-scale Domain Dynamics and Adenosylcobalamin Reorientation Orchestrate Radical Catalysis in Ornithine 4,5-Aminomutase

Wolthers, Kirsten R.; Levy, Colin; Scrutton, Nigel S.; Leys, David
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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d-Ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii converts d-ornithine to 2,4-diaminopentanoic acid by way of radical propagation from an adenosylcobalamin (AdoCbl) to a pyridoxal 5′-phosphate (PLP) cofactor. We have solved OAM crystal structures in different catalytic states that together demonstrate unusual stability of the AdoCbl Co-C bond and that radical catalysis is coupled to large-scale domain motion. The 2.0-Å substrate-free enzyme crystal structure reveals the Rossmann domain, harboring the intact AdoCbl cofactor, is tilted toward the edge of the PLP binding triose-phosphate isomerase barrel domain. The PLP forms an internal aldimine link to the Rossmann domain through Lys629, effectively locking the enzyme in this “open” pre-catalytic conformation. The distance between PLP and 5′-deoxyadenosyl group is 23 Å, and large-scale domain movement is thus required prior to radical catalysis. The OAM crystals contain two Rossmann domains within the asymmetric unit that are unconstrained by the crystal lattice. Surprisingly, the binding of various ligands to OAM crystals (in an oxygen-free environment) leads to transimination in the absence of significant reorientation of the Rossmann domains. In contrast, when performed under aerobic conditions...

First-principle studies of intermolecular and intramolecular catalysis of protonated cocaine

Zhan, Chang-Guo; Deng, Shi-Xian; Skiba, Jaime G.; Hayes, Beth A.; Tschampel, Sarah M.; Shields, George C.; Landry, Donald W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 30/07/2005 Português
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We have performed a series of first-principles electronic structure calculations to examine the reaction pathways and the corresponding free energy barriers for the ester hydrolysis of protonated cocaine in its chair and boat conformations. The calculated free energy barriers for the benzoyl ester hydrolysis of protonated chair cocaine are close to the corresponding barriers calculated for the benzoyl ester hydrolysis of neutral cocaine. However, the free energy barrier calculated for the methyl ester hydrolysis of protonated cocaine in its chair conformation is significantly lower than for the methyl ester hydrolysis of neutral cocaine and for the dominant pathway of the benzoyl ester hydrolysis of protonated cocaine. The significant decrease of the free energy barrier, ~ 4 kcal/mol, is attributed to the intramolecular acid catalysis of the methyl ester hydrolysis of protonated cocaine, because the transition state structure is stabilized by the strong hydrogen bond between the carbonyl oxygen of the methyl ester moiety and the protonated tropane N. The relative magnitudes of the free energy barriers calculated for different pathways of the ester hydrolysis of protonated chair cocaine are consistent with the experimental kinetic data for cocaine hydrolysis under physiologic conditions. Similar intramolecular acid catalysis also occurs for the benzoyl ester hydrolysis of (protonated) boat cocaine in the physiologic condition...

The divalent metal ion in the active site of uteroferrin modulates substrate binding and catalysis

Mitić, Nataša; Hadler, Kieran S.; Gahan, Lawrence R; Hengge, Alvan C.; Schenk, Gerhard
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 26/05/2010 Português
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The purple acid phosphatases (PAP) are binuclear metallohydrolases that catalyze the hydrolysis of a broad range of phosphomonoester substrates. The mode of substrate binding during catalysis and the identity of the nucleophile is subject to debate. Here, we used native Fe3+-Fe2+ pig PAP (uteroferrin; Uf) and its Fe3+-Mn2+ derivative to investigate the effect of metal ion substitution on the mechanism of catalysis. Replacement of the Fe2+ by Mn2+ lowers the reactivity of Uf. However, using stopped-flow measurements it could be shown that this replacement facilitates approximately a ten-fold faster reaction between both substrate and inorganic phosphate with the chromophoric Fe3+ site. These data also indicate that in both metal forms of Uf, phenyl phosphate hydrolysis occurs faster than formation of a μ-1,3 phosphate complex. The slower rate of interaction between substrate and the Fe3+ site relative to catalysis suggests that the substrate is hydrolyzed while coordinated only to the divalent metal ion. The likely nucleophile is a water molecule in the second coordination sphere, activated by a hydroxide terminally coordinated to Fe3+. The faster rates of interaction with the Fe3+ site in the Fe3+-Mn2+ derivative than the native Fe3+-Fe2+ form are likely mediated via a hydrogen bond network connecting the first and second coordination spheres...

Direct asymmetric vinylogous Michael addition of cyclic enones to nitroalkenes via dienamine catalysis

Bencivenni, Giorgio; Galzerano, Patrizia; Mazzanti, Andrea; Bartoli, Giuseppe; Melchiorre, Paolo
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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In spite of the many catalytic methodologies available for the asymmetric functionalization of carbonyl compounds at their α and β positions, little progress has been achieved in the enantioselective carbon–carbon bond formation γ to a carbonyl group. Here, we show that primary amine catalysis provides an efficient way to address this synthetic issue, promoting vinylogous nucleophilicity upon selective activation of unmodified cyclic α,β-unsaturated ketones. Specifically, we document the development of the unprecedented direct and vinylogous Michael addition of β-substituted cyclohexenone derivatives to nitroalkenes proceeding under dienamine catalysis. Besides enforcing high levels of diastereo- and enantioselectivity, chiral primary amine catalysts derived from natural cinchona alkaloids ensure complete γ-site selectivity: The resulting, highly functionalized vinylogous Michael adducts, having two stereocenters at the γ and δ positions, are synthesized with very high fidelity. Finally, we describe the extension of the dienamine catalysis-induced vinylogous nucleophilicity to the asymmetric γ-amination of cyclohexene carbaldehyde.

Substituent Effects on Electrophilic Catalysis by the Carbonyl Group: Anatomy of the Rate Acceleration for PLP-Catalyzed Deprotonation of Glycine

Crugeiras, Juan; Rios, Ana; Riveiros, Enrique; Richard, John P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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First-order rate constants, determined by 1H NMR, are reported for deuterium exchange between solvent D2O and the α-amino carbon of glycine in the presence of increasing concentrations of carbonyl compounds (acetone, benzaldehyde and salicylaldehyde) and at different pD and buffer concentrations. These rate data were combined with 1H NMR data that define the position of the equilibrium for formation of imines/iminium ions from addition of glycine to the respective carbonyl compounds, to give second-order rate constants kDO for deprotonation of α-imino carbon by DO−. The assumption that these second-order rate constants lie on linear structure-reactivity correlations between log kOL and pKa was made in estimating the following pKas for deprotonation of α-imino carbon: pKa = 22, glycine–acetone iminium ion; pKa = 27, glycine–benzaldehyde imine; pKa ≈ 23, glycine–benzaldehyde iminium ion; and, pKa = 25, glycine–salicylaldehyde iminium ion. The much lower pKa of 17 [Toth, K.; Richard, J. P. J. Am. Chem. Soc. 2007, 129, 3013–3021] for carbon deprotonation of the adduct between 5′-deoxypyridoxal (DPL) and glycine shows that the strongly electron-withdrawing pyridinium ion is unique in driving the extended delocalization of negative charge from the α-iminium to the α-pyridinium carbon. This favors carbanion protonation at the α–pyridinium carbon...

A Spring-loaded Release Mechanism Regulates Domain Movement and Catalysis in Phosphoglycerate Kinase*

Zerrad, Louiza; Merli, Angelo; Schröder, Gunnar F.; Varga, Andrea; Gráczer, Éva; Pernot, Petra; Round, Adam; Vas, Mária; Bowler, Matthew W.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Phosphoglycerate kinase (PGK) is the enzyme responsible for the first ATP-generating step of glycolysis and has been implicated extensively in oncogenesis and its development. Solution small angle x-ray scattering (SAXS) data, in combination with crystal structures of the enzyme in complex with substrate and product analogues, reveal a new conformation for the resting state of the enzyme and demonstrate the role of substrate binding in the preparation of the enzyme for domain closure. Comparison of the x-ray scattering curves of the enzyme in different states with crystal structures has allowed the complete reaction cycle to be resolved both structurally and temporally. The enzyme appears to spend most of its time in a fully open conformation with short periods of closure and catalysis, thereby allowing the rapid diffusion of substrates and products in and out of the binding sites. Analysis of the open apoenzyme structure, defined through deformable elastic network refinement against the SAXS data, suggests that interactions in a mostly buried hydrophobic region may favor the open conformation. This patch is exposed on domain closure, making the open conformation more thermodynamically stable. Ionic interactions act to maintain the closed conformation to allow catalysis. The short time PGK spends in the closed conformation and its strong tendency to rest in an open conformation imply a spring-loaded release mechanism to regulate domain movement...