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Nigel S. Scrutton - One of the best experts on this subject based on the ideXlab platform.
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driving force analysis of proton tunnelling across a reactivity series for an Enzyme Substrate complex
ChemBioChem, 2008Co-Authors: Parvinder Hothi, Anna Roujeinikova, Paul M Cullis, Michael J Sutcliffe, David Leys, Nigel S. ScruttonAbstract:Quantitative structure-activity relationships are widely used to probe C-H bond breakage by quinoprotein Enzymes. However, we showed recently that p-substituted benzylamines are poor reactivity probes for the quinoprotein aromatic amine dehydrogenase (AADH) because of a requirement for structural change in the Enzyme-Substrate complex prior to C-H bond breakage. This rearrangement is partially rate limiting, which leads to deflated kinetic isotope effects for p-substituted benzylamines. Here we report reactivity (driving force) studies of AADH with p-substituted phenylethylamines for which the kinetic isotope effect (approximately 16) accompanying C-H/C-(2)H bond breakage is elevated above the semi-classical limit. We show bond breakage occurs by quantum tunnelling and that within the context of the environmentally coupled framework for H-tunnelling the presence of the p-substituent places greater demand on the apparent need for fast promoting motions. The crystal structure of AADH soaked with phenylethylamine or methoxyphenylethylamine indicates that the structural change identified with p-substituted benzylamines should not limit the reaction with p-substituted phenylethylamines. This is consistent with the elevated kinetic isotope effects measured with p-substituted phenylethylamines. We find a good correlation in the rate constant for proton transfer with bond dissociation energy for the reactive C-H bond, consistent with a rate that is limited by a Marcus-like tunnelling mechanism. As the driving force becomes larger, the rate of proton transfer increases while the Marcus activation energy becomes smaller. This is the first experimental report of the driving force perturbation of H-tunnelling in Enzymes using a series of related Substrates. Our study provides further support for proton tunnelling in AADH.
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conformational events during ternary Enzyme Substrate complex formation are rate limiting in the catalytic cycle of the light driven Enzyme protochlorophyllide oxidoreductase
Biochemistry, 2008Co-Authors: Derren J Heyes, Michiyo Sakuma, Binuraj R K Menon, Nigel S. ScruttonAbstract:The light-driven Enzyme, protochlorophyllide oxidoreductase (POR), has proven to be an excellent model system for studying the role of protein motions during catalysis. POR catalyzes the trans addition of hydrogen across the C17-C18 double bond of protochlorophyllide (Pchlide), which is a key step in chlorophyll biosynthesis. While we currently have a detailed understanding of the initial photochemical events and the subsequent hydrogen transfer reactions, there remains a lack of information about the slower Substrate binding events leading to the formation of the catalytically active ternary complex. As POR is light-activated, it is relatively straightforward to isolate the ternary Enzyme-Substrate complex in the dark prior to catalysis, which has facilitated the use of a variety of spectroscopic and kinetic probes to study the binding of both Substrates. Herein, we provide a detailed kinetic and thermodynamic description of these processes and show that the binding events are complex, involving multiple conformational states en route to the formation of a ternary complex that is primed for photoactivation. The initial binding of NADPH involves three distinct steps, which appear to be necessary for the optimal alignment of the cofactor in the Enzyme active site. This is followed by the binding of the Pchlide Substrate and subsequent Substrate-induced conformational changes within the Enzyme that occur prior to the formation of the final "poised" conformational state. These studies, which provide important information on the formation of the reactive conformation, reveal that ternary complex formation is the rate-limiting step in the overall reaction and is controlled by slow conformational changes in the protein.
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the ph dependence of kinetic isotope effects in monoamine oxidase a indicates stabilization of the neutral amine in the Enzyme Substrate complex
FEBS Journal, 2008Co-Authors: Rachel V Dunn, Ker R Marshall, Andrew W Munro, Nigel S. ScruttonAbstract:A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine Substrate in the Enzyme–Substrate complex. However, recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the suggestion that it is the protonated form of the amine Substrate that binds to the Enzyme. To investigate this further, the pH dependence of monoamine oxidase A was characterized by both steady-state and stopped-flow techniques with protiated and deuterated Substrates. For all Substrates used, there is a macroscopic ionization in the Enzyme–Substrate complex attributed to a deprotonation event required for optimal catalysis with a pKa of 7.4–8.4. In stopped-flow assays, the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH, leading to assignment of this catalytically important deprotonation to that of the bound amine Substrate. The acid limb of the bell-shaped pH profile for the rate of flavin reduction over the Substrate binding constant (kred/Ks, reporting on ionizations in the free Enzyme and/or free Substrate) is due to deprotonation of the free Substrate, and the alkaline limb is due to unfavourable deprotonation of an unknown group on the Enzyme at high pH. The pKa of the free amine is above 9.3 for all Substrates, and is greatly perturbed (ΔpKa∼ 2) on binding to the Enzyme active site. This perturbation of the Substrate amine pKa on binding to the Enzyme has been observed with other amine oxidases, and likely identifies a common mechanism for increasing the effective concentration of the neutral form of the Substrate in the Enzyme–Substrate complex, thus enabling efficient functioning of these Enzymes at physiologically relevant pH.
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The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the Enzyme-Substrate complex.
The FEBS journal, 2008Co-Authors: Rachel V Dunn, Ker R Marshall, Andrew W Munro, Nigel S. ScruttonAbstract:A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine Substrate in the Enzyme-Substrate complex. However, recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the suggestion that it is the protonated form of the amine Substrate that binds to the Enzyme. To investigate this further, the pH dependence of monoamine oxidase A was characterized by both steady-state and stopped-flow techniques with protiated and deuterated Substrates. For all Substrates used, there is a macroscopic ionization in the Enzyme-Substrate complex attributed to a deprotonation event required for optimal catalysis with a pK(a) of 7.4-8.4. In stopped-flow assays, the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH, leading to assignment of this catalytically important deprotonation to that of the bound amine Substrate. The acid limb of the bell-shaped pH profile for the rate of flavin reduction over the Substrate binding constant (k(red)/K(s), reporting on ionizations in the free Enzyme and/or free Substrate) is due to deprotonation of the free Substrate, and the alkaline limb is due to unfavourable deprotonation of an unknown group on the Enzyme at high pH. The pK(a) of the free amine is above 9.3 for all Substrates, and is greatly perturbed (DeltapK(a) approximately 2) on binding to the Enzyme active site. This perturbation of the Substrate amine pK(a) on binding to the Enzyme has been observed with other amine oxidases, and likely identifies a common mechanism for increasing the effective concentration of the neutral form of the Substrate in the Enzyme-Substrate complex, thus enabling efficient functioning of these Enzymes at physiologically relevant pH.
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reductive half reaction of the h172q mutant of trimethylamine dehydrogenase evidence against a carbanion mechanism and assignment of kinetically influential ionizations in the Enzyme Substrate complex
Biochemical Journal, 1999Co-Authors: Jaswir Basran, Michael J Sutcliffe, Russ Hille, Nigel S. ScruttonAbstract:The reactions of wild-type trimethylamine dehydrogenase (TMADH) and of a His-172-->Gln (H172Q) mutant were studied by rapid-mixing stopped-flow spectroscopy over the pH range 6.0-10.5, to address the potential role of His-172 in abstracting a proton from the Substrate in a 'carbanion' mechanism for C-H bond cleavage. The pH-dependence of the limiting rate for flavin reduction (klim) was studied as a function of pH for the wild-type Enzyme with perdeuterated trimethylamine as Substrate. The use of perdeuterated trimethylamine facilitated the unequivocal identification of two kinetically influential ionizations in the Enzyme-Substrate complex, with macroscopic pKa values of 6.5+/-0.2 and 8.4+/-0.1. A plot of klim/Kd revealed a bell-shaped curve and two kinetically influential ionizations with macroscopic pKa values of 9.4+/-0.1 and 10.5+/-0.1. Mutagenesis of His-172, a potential active-site base and a component of a novel Tyr-His-Asp triad in the active site of TMADH, revealed that the pKa of 8.4+/-0.1 for the wild-type Enzyme-Substrate complex represents ionization of the imidazolium side-chain of His-172. H172Q TMADH retains catalytic competence throughout the pH range investigated. At pH 10.5, and in contrast with the wild-type Enzyme, flavin reduction in H172Q TMADH is biphasic. The fast phase is dependent on the trimethylamine concentration and exhibits a kinetic isotope effect of about 3; C-H bond cleavage is thus partially rate-limiting. In contrast, the slow phase does not show hyperbolic dependence on Substrate concentration, and the observed rate shows no dependence on isotope, revealing that C-H bond cleavage is not rate-limiting. The analysis of H172Q TMADH, together with data recently acquired for the Y169F mutant of TMADH, reveals that C-H bond breakage is not initiated via abstraction of a proton from the Substrate by an active-site base. The transfer of reducing equivalents to flavin via a carbanion mechanism is therefore unlikely.
Arti Dua - One of the best experts on this subject based on the ideXlab platform.
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parallel versus off pathway michaelis menten mechanism for single Enzyme kinetics of a fluctuating Enzyme
arXiv: Chemical Physics, 2015Co-Authors: Ashutosh Kumar, Hiranmay Maity, Arti DuaAbstract:Recent fluorescence spectroscopy measurements of the turnover time distribution of single-Enzyme turnover kinetics of $\beta$-galactosidase provide evidence of Michaelis-Menten kinetics at low Substrate concentration. However, at high Substrate concentrations, the dimensionless variance of the turnover time distribution shows systematic deviations from the Michaelis-Menten prediction. This difference is attributed to conformational fluctuations in both the Enzyme and the Enzyme-Substrate complex and to the possibility of both parallel and off-pathway kinetics. Here, we use the chemical master equation to model the kinetics of a single fluctuating Enzyme that can yield a product through either parallel or off-pathway mechanisms. An exact expression is obtained for the turnover time distribution from which the mean turnover time and randomness parameters are calculated. The parallel and off-pathway mechanisms yield strikingly different dependences of the mean turnover time and the randomness parameter on the Substrate concentration. In the parallel mechanism, the distinct contributions of Enzyme and Enzyme-Substrate fluctuations are clearly discerned from the variation of the randomness parameter with Substrate concentration. From these general results we conclude that an off-pathway mechanism, with substantial Enzyme-Substrate fluctuations, is needed to rationalize the experimental findings of single-Enzyme turnover kinetics of $\beta$-galactosidase.
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Parallel versus Off-Pathway Michaelis-Menten Mechanism for Single-Enzyme Kinetics of a Fluctuating Enzyme
Journal of Physical Chemistry B, 2015Co-Authors: Ashutosh Kumar, Hiranmay Maity, Arti DuaAbstract:Recent fluorescence spectroscopy measurements of the turnover time distribution of single-Enzyme turnover kinetics of β-galactosidase provide evidence of Michaelis-Menten kinetics at low Substrate concentration. However, at high Substrate concentrations, the dimensionless variance of the turnover time distribution shows systematic deviations from the Michaelis-Menten prediction. This difference is attributed to conformational fluctuations in both the Enzyme and the Enzyme-Substrate complex and to the possibility of both parallel- and off-pathway kinetics. Here, we use the chemical master equation to model the kinetics of a single fluctuating Enzyme that can yield a product through either parallel- or off-pathway mechanisms. An exact expression is obtained for the turnover time distribution from which the mean turnover time and randomness parameters are calculated. The parallel- and off-pathway mechanisms yield strikingly different dependences of the mean turnover time and the randomness parameter on the Substrate concentration. In the parallel mechanism, the distinct contributions of Enzyme and Enzyme-Substrate fluctuations are clearly discerned from the variation of the randomness parameter with Substrate concentration. From these general results, we conclude that an off-pathway mechanism, with substantial Enzyme-Substrate fluctuations, is needed to rationalize the experimental findings of single-Enzyme turnover kinetics of β-galactosidase.
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parallel versus off pathway michaelis menten mechanism for single Enzyme kinetics of a fluctuating Enzyme b
The Journal of Physical Chemistry, 2015Co-Authors: Ashutosh Kumar, Hiranmay Maity, Arti DuaAbstract:Recent fluorescence spectroscopy measurements of the turnover time distribution of single-Enzyme turnover kinetics of β-galactosidase provide evidence of Michaelis–Menten kinetics at low Substrate concentration. However, at high Substrate concentrations, the dimensionless variance of the turnover time distribution shows systematic deviations from the Michaelis–Menten prediction. This difference is attributed to conformational fluctuations in both the Enzyme and the Enzyme–Substrate complex and to the possibility of both parallel- and off-pathway kinetics. Here, we use the chemical master equation to model the kinetics of a single fluctuating Enzyme that can yield a product through either parallel- or off-pathway mechanisms. An exact expression is obtained for the turnover time distribution from which the mean turnover time and randomness parameters are calculated. The parallel- and off-pathway mechanisms yield strikingly different dependences of the mean turnover time and the randomness parameter on the Substrate concentration. In the parallel mechanism, the distinct contributions of Enzyme and Enzyme–Substrate fluctuations are clearly discerned from the variation of the randomness parameter with Substrate concentration. From these general results, we conclude that an off-pathway mechanism, with substantial Enzyme–Substrate fluctuations, is needed to rationalize the experimental findings of single-Enzyme turnover kinetics of β-galactosidase.
Douglas A Mitchell - One of the best experts on this subject based on the ideXlab platform.
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cell free biosynthesis to evaluate lasso peptide formation and Enzyme Substrate tolerance
Journal of the American Chemical Society, 2021Co-Authors: Ashley M Kretsch, Laura M Daigh, Mark J Burk, Douglas A MitchellAbstract:Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like, threaded conformation. Owing to a locked three-dimensional structure, lasso peptides can be unusually stable toward heat and proteolytic degradation. Some lasso peptides have been shown to bind human cell-surface receptors and exhibit anticancer properties, while others display antibacterial or antiviral activities. All known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent class of RiPPs; however, the discovery, isolation, and characterization of lasso peptides are constrained by the lack of an efficient production system. In this study, we employ a cell-free biosynthesis (CFB) strategy to address longstanding challenges associated with lasso peptide production. We report the successful use of CFB for the formation of an array of sequence-diverse lasso peptides that include known examples as well as a new predicted lasso peptide from Thermobifida halotolerans. We further demonstrate the utility of CFB to rapidly generate and characterize multisite precursor peptide variants to evaluate the Substrate tolerance of the biosynthetic pathway. By evaluating more than 1000 randomly chosen variants, we show that the lasso-forming cyclase from the fusilassin pathway is capable of producing millions of sequence-diverse lasso peptides via CFB. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.
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cell free biosynthesis to evaluate lasso peptide formation and Enzyme Substrate tolerance
bioRxiv, 2020Co-Authors: Ashley M Kretsch, Laura M Daigh, Mark J Burk, Douglas A MitchellAbstract:Abstract Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like structure. Owing to a rigid topology, lasso peptides are unusually stable towards heat and proteolytic degradation. Some lasso peptides have been shown to bind human cell-surface receptors and exhibit anticancer properties, while others display antibacterial or antiviral activities. Known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent RiPP class; however, the discovery, isolation, and characterization of lasso peptides are constrained by the lack of an efficient production system. In this study, we employ a cell-free biosynthesis (CFB) strategy to address the longstanding challenges associated with lasso peptide production. We report the successful formation of a diverse array of lasso peptides that include known examples as well as a new predicted lasso peptide from Thermobifida halotolerans. We further demonstrate the utility of CFB to rapidly generate and characterize multisite precursor peptide variants in order to evaluate the Substrate tolerance of the biosynthetic pathway. We show that the lasso-forming cyclase from the fusilassin pathway can produce millions of sequence-diverse lasso peptides via CFB with an extraordinary level of sequence permissiveness within the ring region of the lasso peptide. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.
Jianjun Zhang - One of the best experts on this subject based on the ideXlab platform.
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a series of niii flavonolate complexes as structural and functional es Enzyme Substrate models of the niii containing quercetin 2 3 dioxygenase
Dalton Transactions, 2014Co-Authors: Yingji Sun, Qianqian Huang, Jianjun ZhangAbstract:NiII-flavonolate complexes [NiIILR(fla)] (LRH: 2-{[bis(pyridin-2-ylmethyl)amino]methyl}-p/m-R-benzoic acid, R: p-OMe (1), p-Me (2), m-Br (4) and m-NO2 (5), fla: flavonolate) were synthesized and characterized with relevance to structural and functional models for the ES (Enzyme–Substrate) adduct of the NiII-containing quercetin 2,3-dioxygenase (2,3-QD). Their structures, spectroscopic features, redox properties and the reactivity toward molecular oxygen have been investigated. The complexes show a similar distorted octahedral structure and higher Enzyme-type dioxygenation reactivity than other reported metal-flavonolate complexes in the oxidative O-heterocyclic ring-opening of the bound Substrate flavonolate at lower temperature owing to the introduced carboxylate group in the supporting model ligands. The reaction rate shows first-order dependence on both of the complex and O2 and the second-order rate constant k fits a Hammett linear free energy relationship (ρ = −0.71) for the substituent group in the supporting model ligand LR. The complexes exhibit substituent group dependent structures, properties and reactivity and there are some relationship among them, which could be ascribed to the electronic nature of the substituent group via the benzoate, NiII ion and O(4)C(27)–C(21)C(22) “electron conduit”. In a word, the stronger electron donating group could induce a smaller torsion angle, larger λmax and lower redox potential of the bound flavonolate, making a higher reactivity finally. This study is the first example of a series of structural and functional ES models of the NiII-containing 2,3-QD, providing important insights into the structure–property–reactivity relationship, the electronic substituent effects and carboxylate effects on the enzymatic reactivity and the catalytic role of the NiII-containing 2,3-QD.
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series of structural and functional models for the es Enzyme Substrate complex of the co ii containing quercetin 2 3 dioxygenase
Inorganic Chemistry, 2014Co-Authors: Qianqian Huang, Jianjun ZhangAbstract:A series of mononuclear CoII–flavonolate complexes [CoIILR(fla)] (LRH = 2-{[bis(pyridin-2-ylmethyl)amino]methyl}-p/m-R-benzoic acid; R = p-OMe (1), p-Me (2), m-Br (4), and m-NO2 (5); fla = flavonolate) were designed and synthesized as structural and functional models for the ES (Enzyme–Substrate) complexes to mimic the active site of the Co(II)-containing quercetin 2,3-dioxygenase (Co–2,3-QD). The metal center Co(II) ion in each complex shows a similar distorted octahedral geometry. The model complexes display high Enzyme-type dioxygenation reactivity (oxidative O-heterocyclic ring opening of the coordinated Substrate flavonolate) at low temperature, presumably due to the attached carboxylate group in the ligands. The reactivity exhibits a substituent group dependent order of −OMe (1) > −Me (2) > −H (3)14b > −Br (4) > −NO2 (5), and the Hammett plot is linear (ρ = −0.78). This can be explained as the electronic nature of the substituent group in the ligands may influence the conformation and redox potentia...
Qianqian Huang - One of the best experts on this subject based on the ideXlab platform.
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a series of niii flavonolate complexes as structural and functional es Enzyme Substrate models of the niii containing quercetin 2 3 dioxygenase
Dalton Transactions, 2014Co-Authors: Yingji Sun, Qianqian Huang, Jianjun ZhangAbstract:NiII-flavonolate complexes [NiIILR(fla)] (LRH: 2-{[bis(pyridin-2-ylmethyl)amino]methyl}-p/m-R-benzoic acid, R: p-OMe (1), p-Me (2), m-Br (4) and m-NO2 (5), fla: flavonolate) were synthesized and characterized with relevance to structural and functional models for the ES (Enzyme–Substrate) adduct of the NiII-containing quercetin 2,3-dioxygenase (2,3-QD). Their structures, spectroscopic features, redox properties and the reactivity toward molecular oxygen have been investigated. The complexes show a similar distorted octahedral structure and higher Enzyme-type dioxygenation reactivity than other reported metal-flavonolate complexes in the oxidative O-heterocyclic ring-opening of the bound Substrate flavonolate at lower temperature owing to the introduced carboxylate group in the supporting model ligands. The reaction rate shows first-order dependence on both of the complex and O2 and the second-order rate constant k fits a Hammett linear free energy relationship (ρ = −0.71) for the substituent group in the supporting model ligand LR. The complexes exhibit substituent group dependent structures, properties and reactivity and there are some relationship among them, which could be ascribed to the electronic nature of the substituent group via the benzoate, NiII ion and O(4)C(27)–C(21)C(22) “electron conduit”. In a word, the stronger electron donating group could induce a smaller torsion angle, larger λmax and lower redox potential of the bound flavonolate, making a higher reactivity finally. This study is the first example of a series of structural and functional ES models of the NiII-containing 2,3-QD, providing important insights into the structure–property–reactivity relationship, the electronic substituent effects and carboxylate effects on the enzymatic reactivity and the catalytic role of the NiII-containing 2,3-QD.
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series of structural and functional models for the es Enzyme Substrate complex of the co ii containing quercetin 2 3 dioxygenase
Inorganic Chemistry, 2014Co-Authors: Qianqian Huang, Jianjun ZhangAbstract:A series of mononuclear CoII–flavonolate complexes [CoIILR(fla)] (LRH = 2-{[bis(pyridin-2-ylmethyl)amino]methyl}-p/m-R-benzoic acid; R = p-OMe (1), p-Me (2), m-Br (4), and m-NO2 (5); fla = flavonolate) were designed and synthesized as structural and functional models for the ES (Enzyme–Substrate) complexes to mimic the active site of the Co(II)-containing quercetin 2,3-dioxygenase (Co–2,3-QD). The metal center Co(II) ion in each complex shows a similar distorted octahedral geometry. The model complexes display high Enzyme-type dioxygenation reactivity (oxidative O-heterocyclic ring opening of the coordinated Substrate flavonolate) at low temperature, presumably due to the attached carboxylate group in the ligands. The reactivity exhibits a substituent group dependent order of −OMe (1) > −Me (2) > −H (3)14b > −Br (4) > −NO2 (5), and the Hammett plot is linear (ρ = −0.78). This can be explained as the electronic nature of the substituent group in the ligands may influence the conformation and redox potentia...
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flavonolate complexes of mii m mn fe co ni cu and zn structural and functional models for the es Enzyme Substrate complex of quercetin 2 3 dioxygenase
Inorganic Chemistry, 2013Co-Authors: Qianqian Huang, Tetsuro Tano, Shinobu ItohAbstract:A series of flavonolate complexes [MIIL(fla)] (M = Mn (1), Fe (2), Co (3), Ni (4), Cu (5), and Zn (6), LH: 2-{[bis(pyridin-2-ylmethyl)amino]methyl}benzoic acid, fla: flavonolate) have been synthesized as structural and functional models for the ES (Enzyme–Substrate) complexes of the active site of various MII-containing quercetin 2,3-dioxygenase (2,3-QD) and their structures, spectroscopic features, and redox properties, as well as the reactivity toward molecular oxygen, have been investigated. The metal centers of [FeIIL(fla)]·H2O (2), [CoIIL(fla)]·CH3OH (3), and [NiIIL(fla)] (4) exhibit a distorted octahedral geometry with two oxygen atoms of fla, one oxygen atom of the benzoate group of ligand L, and three nitrogen atoms of ligand L, in which oxygen atom of the carbonyl group of fla and one of the pyridine nitrogen atoms occupy the axial positions. The complexes [MIIL(fla)] exhibit relatively high reactivity in the oxidative ring-opening of the bound flavonolate at lower temperature, presumably due to ...