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Troels Koch - One of the best experts on this subject based on the ideXlab platform.
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stopped flow Kinetics of locked nucleic acid lna oligonucleotide duplex formation studies of lna dna and dna dna interactions
Biochemical Journal, 2001Co-Authors: Ulla Christensen, Nana Jacobsen, Vivek K Rajwanshi, Jesper Wengel, Troels KochAbstract:The locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2′- O ,4′- C -methylene bridge added to the ribose ring. Oligonucleotides that contain LNA monomers have shown greatly enhanced thermal stability when hybridized to complementary DNA and RNA and are considered most promising candidates for efficient recognition of a given mixed sequence in a nucleic acid duplex and as an antisense molecule. Here the Kinetics and thermodynamics of a series of oligonucleotide duplex formations of DNA–DNA and DNA–LNA octamers were studied using Stopped-Flow absorption measurements at 25°C and melting curves. The reactions of the DNA octamer 5′-CAGGAGCA-3′ with its complementary DNA octamer 5′-TGCTCCTG-3′, and with the LNA octamers 5′- T L GCTCCTG-3′ (LNA-1), 5′- T L GC T L CCTG-3′ (LNA-2) and 5′- T L GC T L CC T L G-3′(LNA-3), containing respectively one, two or three thymidine 2′- O ,4′- C -methylene-(D-ribofuranosyl) nucleotide monomers, designated T L , were studied. In all cases were seen fast second-order association reactions with k obs = 2×10 7 M -1 ˙s -1 . At 25°C the dissociation constants of the duplexes obtained from melting curves were: DNA–DNA, 10nM; DNA–LNA-1, 20nM; DNA–LNA-2, 2nM; and DNA–LNA-3, 0.3nM; thus the greatly enhanced duplex stability induced by LNA is confirmed. Since the association rates were all equal this increase in stability is due to slower rates of dissociation of the complexes.
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stopped flow Kinetics of locked nucleic acid lna oligonucleotide duplex formation studies of lna dna and dna dna interactions
Biochemical Journal, 2001Co-Authors: Ulla Christensen, Nana Jacobsen, Vivek K Rajwanshi, Jesper Wengel, Troels KochAbstract:The locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2'-O,4'-C-methylene bridge added to the ribose ring. Oligonucleotides that contain LNA monomers have shown greatly enhanced thermal stability when hybridized to complementary DNA and RNA and are considered most promising candidates for efficient recognition of a given mixed sequence in a nucleic acid duplex and as an antisense molecule. Here the Kinetics and thermodynamics of a series of oligonucleotide duplex formations of DNA-DNA and DNA-LNA octamers were studied using Stopped-Flow absorption measurements at 25 degrees C and melting curves. The reactions of the DNA octamer 5'-CAGGAGCA-3' with its complementary DNA octamer 5'-TGCTCCTG-3', and with the LNA octamers 5'-T(L)GCTCCTG-3' (LNA-1), 5'-T(L)GCT(L)CCTG-3' (LNA-2) and 5'-T(L)GCT(L)CCT(L)G-3'(LNA-3), containing respectively one, two or three thymidine 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotide monomers, designated T(L), were studied. In all cases were seen fast second-order association reactions with k(obs)=2x10(7) M(-1)s(-1). At 25 degrees C the dissociation constants of the duplexes obtained from melting curves were: DNA-DNA, 10 nM; DNA-LNA-1, 20 nM; DNA-LNA-2, 2 nM; and DNA-LNA-3, 0.3 nM; thus the greatly enhanced duplex stability induced by LNA is confirmed. Since the association rates were all equal this increase in stability is due to slower rates of dissociation of the complexes.
Paul A Lindahl - One of the best experts on this subject based on the ideXlab platform.
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Kinetics of co insertion and acetyl group transfer steps and a model of the acetyl coa synthase catalytic mechanism
Journal of the American Chemical Society, 2006Co-Authors: Xiangshi Tan, Ivan V Surovtsev, Paul A LindahlAbstract:Acetyl-CoA synthases/carbon monoxide dehydrogenases are found in homoacetogenic bacteria, methanogenic archaea, and CO-utilizing hydrogenogenic bacteria.1–4 These O2-sensitive bifunctional enzymes allow such organisms to grow chemo-autotrophically on simple inorganic compounds. The enzyme from the homoacetogen Moorella thermoacetica (ACS/CODH) has been studied most extensively.5,6 The β subunits of this 310 kDa α2β2 tetramer catalyze the reversible reduction of CO2 to CO, while the α subunits catalyze the synthesis of acetyl-CoA from CO, CoA, and a methyl group donated from a corrinoid-iron sulfur protein (CoFeSP). At a buffered pH, this is represented by reaction 1. CO+CoA+CH3−Co3+FeSP⇄CH3−C(O)−CoA+Co1+FeSPKACS [1] The active-site for this reaction, called the A-cluster, consists of an [Fe4S4] cubane bridged via a cysteine residue to a Ni ion called proximal Nip. This Ni is also bridged (via 2 other cysteines) to a second Ni ion (distal Nid); thus, Nip is coordinated to 3 bridging thiolates. Nid has an N2S2 square-planar environment including coordination to two amide nitrogens derived from the protein backbone.7–9 Evidence suggests that CO and methyl groups bind to Nip during catalysis.10, 11 Although aspects of the ACS catalytic mechanism remain uncertain, our understanding of it is gradually improving. The components of the A-cluster in its most oxidized redox state (called Aox) appear to be in the {[Fe4S4]2+ Nip2+ Nid2+} electronic configuration.12, 13 This state is inactive for both catalysis and methyl group transfer but it can be activated by a 2-electron reduction corresponding to an apparent midpoint potential of ca. −540 mV vs. NHE at neutral pH.14 The resulting reductively-activated state apparently has the cubane and Nid in the 2+ states, suggesting the unprecedented {[Fe4S4]2+ Nip0 Nid2+} configuration.12,13,14 Although the occurrence of a zero-valent Ni atom in the reductively activated state is not established, we will use this nomenclature throughout in this paper, for convenience if for no other reason. For a full discussion of this issue, readers are referred to the literature.4,13–15 If preferred, “Ni0” can be viewed simply as an electron-counting formalism indicating the Aox state to which 2e− have been added. This formal view does not complicate or bias any interpretation, analysis, or conclusion presented here. Whether the methyl group or CO bind first to the enzyme remains contentious, and reasonable arguments have been made for both cases.5,6,11,16 The one-electron-reduced and CO-bound state of the A-cluster (the S = ½ Ared-CO state) has been proposed to be an intermediate of catalysis as well as an inhibitory state. Recent evidence that ACS/CODH need not pass through the Ared-CO state during catalysis and evidence that reductive activation requires 2 electrons14 compel us to favor the case where the methyl group binds first. It is also known that the reductively activated state accepts a methyl group in the absence of CO, as shown in reaction 2. Ni0+CH3−Co3+FeSP⇄Ni2+−CH3+Co1+FeSPKmet=k+metk−met [2] The resulting methylated state is stable and has been characterized.11,14,17,18 When exposed to CO, e.g. during catalysis, CO is thought to insert into the Ni-methyl bond in accordance with reaction 3. Ni2+−CH3+CO⇄Ni2+−C(O)CH3Kins=k+insk−ins [3] Reaction of the acetyl intermediate with CoA affords acetyl-CoA and regenerates the reductively-activated state, reaction 4. Ni2+−C(O)CH3+CoA⇄CH3C(O)−CoA+Ni0KCoA=k+CoAk−CoA. [4] Reactions 2 – 4 complete the catalytic cycle for the synthesis of acetyl-CoA. Of these steps, only methyl group transfer, reaction 2, has been studied specifically.19–21 In a Stopped-Flow study, ACS/CODH and Ti3+citrate were preincubated to generate the Ni0 state, and then reacted against CH3-Co3+FeSP (also preincubated in Ti3+citrate) and monitored at 390 nm where the product Co1+FeSP absorbs. Under these conditions, the reverse of reaction 2 (i.e. starting from Ni2+-CH3 and Co1+) could barely be detected, indicating that the equilibrium position lies on the products side.21 In contrast, the reverse reaction proceeded rapidly and to near completion when the Ni2+-CH3 was not preincubated with Ti3+citrate. Under these conditions, the equilibrium position appears to be on the reactants side of reaction 2. This reductant-dependent shift in the kinetic and thermodynamic properties of the methyl group transfer reaction is not understood mechanistically. Bhaskar et al. have examined the steady-state Kinetics of the exchange reaction between acetyl-CoA and dephospho-CoA as catalyzed by the ACS/CODH homolog from Methanosarcina barkeri.22 Their results indicate a ping-pong mechanism in which the binding of acetyl-CoA to the enzyme is followed by the release of CoA and formation of the acetyl-intermediate. They proposed that acetyl-CoA binds to the oxidized form of the enzyme, followed by reduction. This was suggested because partially-reduced enzyme exhibited cooperative binding with acetyl-CoA whereas fully reduced enzyme showed simple hyperbolic binding. However, the same behavior would be observed if enzyme were first reduced and then bound with acetyl-CoA. This latter scenario would be congruent with a nucleophilic attack (e.g. by a Ni0 species) on the carbonyl of acetyl-CoA, as in the reverse of reaction 4. The alternative proposal of binding followed by reduction would seem to require attack by Ni2+, a non-nucleophilic metal ion. Using two methods, Bhaskar et al. measured the equilibrium constant for the reverse of reaction 4 to be ~ 0.2 (averaged value),22 suggesting KCoA ~ 5 for a homologous ACS/CODH from a methanogenic archaeon. To date, no direct studies of the CO insertion reaction 3 have been reported, nor have the Kinetics of the reductive elimination of the acetyl group and CoA, reaction 4, been reported. The problem in studying these reactions has been to identify strategies for monitoring them. Using Stopped-Flow Kinetics, we report here that reactions 3 and 4 can be monitored by starting with the methylated state of ACS/CODH. Resulting traces were used to construct a simple kinetic model describing the catalytic mechanism of acetyl-CoA synthase. In this paper we report these results and describe the model.
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function of the tunnel in acetylcoenzyme a synthase carbon monoxide dehydrogenase
Journal of Biological Inorganic Chemistry, 2006Co-Authors: Xiangshi Tan, Anne Volbeda, Juan C Fontecillacamps, Paul A LindahlAbstract:Acetylcoenzyme A synthase/carbon monoxide dehydrogenase (ACS/CODH) contains two Ni-Fe-S active-site clusters (called A and C) connected by a tunnel through which CO and CO2 migrate. Site-directed mutants A578C, L215F, and A219F were designed to block the tunnel at different points along the region between the two C-clusters. Two other mutant proteins F70W and N101Q were designed to block the region that connects the tunnel at the betabeta interface with a water channel also located at that interface. Purified mutant proteins were assayed for Ni/Fe content and examined by electron paramagnetic resonance spectroscopy. Analyses indicate that same metal clusters found in wild-type (WT) ACS/CODH (i.e., the A-, B-, C-, and probably D-clusters) are properly assembled in the mutant enzymes. Stopped-Flow Kinetics revealed that these centers in the mutants are rapidly reducible by dithionite but are only slowly reducible by CO, suggesting an impaired ability of CO to migrate through the tunnel to the C-cluster. Relative to the WT enzyme, mutant proteins exhibited little CODH or ACS activity (using CO2 as a substrate). Some ACS activity was observed when CO was a substrate, but not the cooperative CO inhibition effect characteristic of WT ACS/CODH. These results suggest that CO and CO2 enter and exit the enzyme at the water channel along the betabeta subunit interface. They also suggest two pathways for CO during synthesis of acetylcoenzyme A, including one in which CO enters the enzyme and migrates through the tunnel before binding at the A-cluster, and another in which CO binds the A-cluster directly from the solvent.
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stopped flow Kinetics of methyl group transfer between the corrinoid iron sulfur protein and acetyl coenzyme a synthase from clostridium thermoaceticum
Journal of the American Chemical Society, 2002Co-Authors: Xiangshi Tan, Christopher Sewell, Paul A LindahlAbstract:Kinetics of methyl group transfer between the Ni-Fe-S-containing acetyl-CoA synthase (ACS) and the corrinoid protein (CoFeSP) from Clostridium thermoaceticum were investigated using the Stopped-Flow method at 390 nm. Rates of the reaction CH(3)-Co(3+)FeSP + ACS(red) Co(1+)FeSP + CH(3)-ACS(ox) in both forward and reverse directions were determined using various protein and reductant concentrations. Ti(3+)citrate, dithionite, and CO were used to reductively activate ACS (forming ACS(red)). The simplest mechanism that adequately fit the data involved formation of a [CH(3)-Co(3+)FeSP]:[ACS(red)] complex, methyl group transfer (forming [Co(1+)FeSP]:[CH(3)-ACS(ox)]), product dissociation (forming Co(1+)FeSP + CH(3)-ACS(ox)), and CO binding yielding a nonproductive enzyme state (ACS(red) + CO ACS(red)-CO). Best-fit rate constants were obtained. CO inhibited methyl group transfer by binding ACS(red) in accordance with K(D) = 180 +/- 90 microM. Fits were unimproved when >1 CO was assumed to bind. Ti(3+)citrate and dithionite inhibited the reverse methyl group transfer reaction, probably by reducing the D-site of CH(3)-ACS(ox). This redox site is oxidized by 2e(-) when the methyl cation is transferred from CH(3)-Co(3+)FeSP to ACS(red), and is reduced during the reverse reaction. Best-fit K(D) values for pre- and post-methyl-transfer complexes were 0.12 +/- 0.06 and 0.3 +/- 0.2 microM, respectively. Intracomplex methyl group transfer was reversible with K(eq) = 2.3 +/- 0.9 (k(f)/k(r) = 6.9 s(-1)/3.0 s(-1)). The nucleophilicity of the [Ni(2+)D(red)] unit appears comparable to that of Co(1+) cobalamins. Reduction of the D-site may cause the Ni(2+) of the A-cluster to behave like the Ni of an organometallic Ni(0) complex.
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stopped flow Kinetics of methyl group transfer between the corrinoid iron sulfur protein and acetyl coenzyme a synthase from clostridium thermoaceticum
Journal of the American Chemical Society, 2002Co-Authors: Xiangshi Tan, Christopher Sewell, Paul A LindahlAbstract:Kinetics of methyl group transfer between the Ni−Fe−S-containing acetyl-CoA synthase (ACS) and the corrinoid protein (CoFeSP) from Clostridium thermoaceticum were investigated using the Stopped-Flow method at 390 nm. Rates of the reaction CH3-Co3+FeSP + ACSred ⇄ Co1+FeSP + CH3-ACSox in both forward and reverse directions were determined using various protein and reductant concentrations. Ti3+citrate, dithionite, and CO were used to reductively activate ACS (forming ACSred). The simplest mechanism that adequately fit the data involved formation of a [CH3-Co3+FeSP]:[ACSred] complex, methyl group transfer (forming [Co1+FeSP]:[CH3-ACSox]), product dissociation (forming Co1+FeSP + CH3-ACSox), and CO binding yielding a nonproductive enzyme state (ACSred + CO ⇄ ACSred-CO). Best-fit rate constants were obtained. CO inhibited methyl group transfer by binding ACSred in accordance with KD = 180 ± 90 μM. Fits were unimproved when >1 CO was assumed to bind. Ti3+citrate and dithionite inhibited the reverse methyl group...
Ulla Christensen - One of the best experts on this subject based on the ideXlab platform.
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stopped flow Kinetics of locked nucleic acid lna oligonucleotide duplex formation studies of lna dna and dna dna interactions
Biochemical Journal, 2001Co-Authors: Ulla Christensen, Nana Jacobsen, Vivek K Rajwanshi, Jesper Wengel, Troels KochAbstract:The locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2′- O ,4′- C -methylene bridge added to the ribose ring. Oligonucleotides that contain LNA monomers have shown greatly enhanced thermal stability when hybridized to complementary DNA and RNA and are considered most promising candidates for efficient recognition of a given mixed sequence in a nucleic acid duplex and as an antisense molecule. Here the Kinetics and thermodynamics of a series of oligonucleotide duplex formations of DNA–DNA and DNA–LNA octamers were studied using Stopped-Flow absorption measurements at 25°C and melting curves. The reactions of the DNA octamer 5′-CAGGAGCA-3′ with its complementary DNA octamer 5′-TGCTCCTG-3′, and with the LNA octamers 5′- T L GCTCCTG-3′ (LNA-1), 5′- T L GC T L CCTG-3′ (LNA-2) and 5′- T L GC T L CC T L G-3′(LNA-3), containing respectively one, two or three thymidine 2′- O ,4′- C -methylene-(D-ribofuranosyl) nucleotide monomers, designated T L , were studied. In all cases were seen fast second-order association reactions with k obs = 2×10 7 M -1 ˙s -1 . At 25°C the dissociation constants of the duplexes obtained from melting curves were: DNA–DNA, 10nM; DNA–LNA-1, 20nM; DNA–LNA-2, 2nM; and DNA–LNA-3, 0.3nM; thus the greatly enhanced duplex stability induced by LNA is confirmed. Since the association rates were all equal this increase in stability is due to slower rates of dissociation of the complexes.
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stopped flow Kinetics of locked nucleic acid lna oligonucleotide duplex formation studies of lna dna and dna dna interactions
Biochemical Journal, 2001Co-Authors: Ulla Christensen, Nana Jacobsen, Vivek K Rajwanshi, Jesper Wengel, Troels KochAbstract:The locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2'-O,4'-C-methylene bridge added to the ribose ring. Oligonucleotides that contain LNA monomers have shown greatly enhanced thermal stability when hybridized to complementary DNA and RNA and are considered most promising candidates for efficient recognition of a given mixed sequence in a nucleic acid duplex and as an antisense molecule. Here the Kinetics and thermodynamics of a series of oligonucleotide duplex formations of DNA-DNA and DNA-LNA octamers were studied using Stopped-Flow absorption measurements at 25 degrees C and melting curves. The reactions of the DNA octamer 5'-CAGGAGCA-3' with its complementary DNA octamer 5'-TGCTCCTG-3', and with the LNA octamers 5'-T(L)GCTCCTG-3' (LNA-1), 5'-T(L)GCT(L)CCTG-3' (LNA-2) and 5'-T(L)GCT(L)CCT(L)G-3'(LNA-3), containing respectively one, two or three thymidine 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotide monomers, designated T(L), were studied. In all cases were seen fast second-order association reactions with k(obs)=2x10(7) M(-1)s(-1). At 25 degrees C the dissociation constants of the duplexes obtained from melting curves were: DNA-DNA, 10 nM; DNA-LNA-1, 20 nM; DNA-LNA-2, 2 nM; and DNA-LNA-3, 0.3 nM; thus the greatly enhanced duplex stability induced by LNA is confirmed. Since the association rates were all equal this increase in stability is due to slower rates of dissociation of the complexes.
Olga S Fedorova - One of the best experts on this subject based on the ideXlab platform.
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pre steady state kinetic and structural analysis of interaction of methionine γ lyase from citrobacter freundii with inhibitors
Journal of Biological Chemistry, 2015Co-Authors: Nikita A. Kuznetsov, Olga S Fedorova, Nicolai G Faleev, Alexandra A Kuznetsova, Elena A Morozova, S V Revtovich, Natalya V Anufrieva, Alexei Nikulin, Tatyana V DemidkinaAbstract:Abstract Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the β-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state Stopped-Flow Kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 A resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.
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reversible chemical step and rate limiting enzyme regeneration in the reaction catalyzed by formamidopyrimidine dna glycosylase
Biochemistry, 2009Co-Authors: Nikita A. Kuznetsov, Vladimir V Koval, Dmitry O. Zharkov, Malcolm Buckle, Olga S FedorovaAbstract:Formamidopyrimidine-DNA N-glycosylase (Fpg) operates in the base excision repair pathway in bacteria by removing oxidized guanine bases from DNA and can also cleave the nascent or preformed abasic DNA by β,δ-elimination. In this work, we have used the quench-flow technique (i) to show that the Kinetics of processing of 7,8-dihydro-8-oxoguanine and abasic site lesions by Fpg from Escherichia coli involves a burst phase and a stationary phase, (ii) to establish the reaction kinetic scheme, and (iii) to calculate the rate constants for the reaction steps. A comparison of the quench-flow results with the data from earlier Stopped-Flow Kinetics with tryptophan and 2-aminopurine fluorescence detection reveals that the cleaved product formation is initially reversible; it is followed by conformational changes in the enzyme and DNA molecules that represent the postchemical irreversible rate-limiting steps. We have applied mass spectrometry with electrospray ionization to follow the appearance and disappearance of...
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pre steady state kinetic study of substrate specificity of escherichia coli formamidopyrimidine dna glycosylase
Biochemistry, 2007Co-Authors: Nikita A. Kuznetsov, Yuri N Vorobjev, Vladimir V Koval, Kenneth T. Douglas, Dmitry O. Zharkov, Georgy A Nevinsky, Olga S FedorovaAbstract:Formamidopyrimidine-DNA glycosylase (Fpg) is responsible for removal of 8-oxoguanine (8-oxoG) and other oxidized purine lesions from DNA and can also excise some oxidatively modified pyrimidines [such as dihydrouracil (DHU)]. Fpg is also specific for a base opposite the lesion, efficiently excising 8-oxoG paired with C but not with A. We have applied Stopped-Flow Kinetics using intrinsic tryptophan fluorescence of the enzyme and fluorescence of 2-aminopurine-labeled DNA to analyze the conformational dynamics of Escherichia coli Fpg during processing of good substrates (8-oxoG·C), poor substrates (8-oxoG·A), and substrates of unclear specificity (such as DHU and 8-oxoG opposite T or G). The analysis of fluorescence traces allows us to conclude that when the enzyme encounters its true substrate, 8-oxoG·C, the complex enters the productive catalytic reaction after ∼50 ms, partitioning the substrate away from the competing dissociation process, while poor substrates linger in the initial encounter complex for longer. Several intermediate ES complexes were attributed to different structures that exist along the reaction pathway. A likely sequence of events is that the damaged base is first destabilized by the enzyme binding and then everted from DNA, followed by insertion of several amino acid residues into DNA and isomerization of the enzyme into a pre-excision complex. We conclude that rejection of the incorrect substrates occurs mostly at the early stage of formation of the pre-eversion recognition complex, supporting the role of indirect readout in damage recognition.
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pre steady state kinetic study of substrate specificity of escherichia coli formamidopyrimidine dna glycosylase
Biochemistry, 2007Co-Authors: Nikita A. Kuznetsov, Yuri N Vorobjev, Vladimir V Koval, Kenneth T. Douglas, Dmitry O. Zharkov, Georgy A Nevinsky, Olga S FedorovaAbstract:: Formamidopyrimidine-DNA glycosylase (Fpg) is responsible for removal of 8-oxoguanine (8-oxoG) and other oxidized purine lesions from DNA and can also excise some oxidatively modified pyrimidines [such as dihydrouracil (DHU)]. Fpg is also specific for a base opposite the lesion, efficiently excising 8-oxoG paired with C but not with A. We have applied Stopped-Flow Kinetics using intrinsic tryptophan fluorescence of the enzyme and fluorescence of 2-aminopurine-labeled DNA to analyze the conformational dynamics of Escherichia coli Fpg during processing of good substrates (8-oxoG.C), poor substrates (8-oxoG.A), and substrates of unclear specificity (such as DHU and 8-oxoG opposite T or G). The analysis of fluorescence traces allows us to conclude that when the enzyme encounters its true substrate, 8-oxoG.C, the complex enters the productive catalytic reaction after approximately 50 ms, partitioning the substrate away from the competing dissociation process, while poor substrates linger in the initial encounter complex for longer. Several intermediate ES complexes were attributed to different structures that exist along the reaction pathway. A likely sequence of events is that the damaged base is first destabilized by the enzyme binding and then everted from DNA, followed by insertion of several amino acid residues into DNA and isomerization of the enzyme into a pre-excision complex. We conclude that rejection of the incorrect substrates occurs mostly at the early stage of formation of the pre-eversion recognition complex, supporting the role of indirect readout in damage recognition.
Xiangshi Tan - One of the best experts on this subject based on the ideXlab platform.
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Kinetics of co insertion and acetyl group transfer steps and a model of the acetyl coa synthase catalytic mechanism
Journal of the American Chemical Society, 2006Co-Authors: Xiangshi Tan, Ivan V Surovtsev, Paul A LindahlAbstract:Acetyl-CoA synthases/carbon monoxide dehydrogenases are found in homoacetogenic bacteria, methanogenic archaea, and CO-utilizing hydrogenogenic bacteria.1–4 These O2-sensitive bifunctional enzymes allow such organisms to grow chemo-autotrophically on simple inorganic compounds. The enzyme from the homoacetogen Moorella thermoacetica (ACS/CODH) has been studied most extensively.5,6 The β subunits of this 310 kDa α2β2 tetramer catalyze the reversible reduction of CO2 to CO, while the α subunits catalyze the synthesis of acetyl-CoA from CO, CoA, and a methyl group donated from a corrinoid-iron sulfur protein (CoFeSP). At a buffered pH, this is represented by reaction 1. CO+CoA+CH3−Co3+FeSP⇄CH3−C(O)−CoA+Co1+FeSPKACS [1] The active-site for this reaction, called the A-cluster, consists of an [Fe4S4] cubane bridged via a cysteine residue to a Ni ion called proximal Nip. This Ni is also bridged (via 2 other cysteines) to a second Ni ion (distal Nid); thus, Nip is coordinated to 3 bridging thiolates. Nid has an N2S2 square-planar environment including coordination to two amide nitrogens derived from the protein backbone.7–9 Evidence suggests that CO and methyl groups bind to Nip during catalysis.10, 11 Although aspects of the ACS catalytic mechanism remain uncertain, our understanding of it is gradually improving. The components of the A-cluster in its most oxidized redox state (called Aox) appear to be in the {[Fe4S4]2+ Nip2+ Nid2+} electronic configuration.12, 13 This state is inactive for both catalysis and methyl group transfer but it can be activated by a 2-electron reduction corresponding to an apparent midpoint potential of ca. −540 mV vs. NHE at neutral pH.14 The resulting reductively-activated state apparently has the cubane and Nid in the 2+ states, suggesting the unprecedented {[Fe4S4]2+ Nip0 Nid2+} configuration.12,13,14 Although the occurrence of a zero-valent Ni atom in the reductively activated state is not established, we will use this nomenclature throughout in this paper, for convenience if for no other reason. For a full discussion of this issue, readers are referred to the literature.4,13–15 If preferred, “Ni0” can be viewed simply as an electron-counting formalism indicating the Aox state to which 2e− have been added. This formal view does not complicate or bias any interpretation, analysis, or conclusion presented here. Whether the methyl group or CO bind first to the enzyme remains contentious, and reasonable arguments have been made for both cases.5,6,11,16 The one-electron-reduced and CO-bound state of the A-cluster (the S = ½ Ared-CO state) has been proposed to be an intermediate of catalysis as well as an inhibitory state. Recent evidence that ACS/CODH need not pass through the Ared-CO state during catalysis and evidence that reductive activation requires 2 electrons14 compel us to favor the case where the methyl group binds first. It is also known that the reductively activated state accepts a methyl group in the absence of CO, as shown in reaction 2. Ni0+CH3−Co3+FeSP⇄Ni2+−CH3+Co1+FeSPKmet=k+metk−met [2] The resulting methylated state is stable and has been characterized.11,14,17,18 When exposed to CO, e.g. during catalysis, CO is thought to insert into the Ni-methyl bond in accordance with reaction 3. Ni2+−CH3+CO⇄Ni2+−C(O)CH3Kins=k+insk−ins [3] Reaction of the acetyl intermediate with CoA affords acetyl-CoA and regenerates the reductively-activated state, reaction 4. Ni2+−C(O)CH3+CoA⇄CH3C(O)−CoA+Ni0KCoA=k+CoAk−CoA. [4] Reactions 2 – 4 complete the catalytic cycle for the synthesis of acetyl-CoA. Of these steps, only methyl group transfer, reaction 2, has been studied specifically.19–21 In a Stopped-Flow study, ACS/CODH and Ti3+citrate were preincubated to generate the Ni0 state, and then reacted against CH3-Co3+FeSP (also preincubated in Ti3+citrate) and monitored at 390 nm where the product Co1+FeSP absorbs. Under these conditions, the reverse of reaction 2 (i.e. starting from Ni2+-CH3 and Co1+) could barely be detected, indicating that the equilibrium position lies on the products side.21 In contrast, the reverse reaction proceeded rapidly and to near completion when the Ni2+-CH3 was not preincubated with Ti3+citrate. Under these conditions, the equilibrium position appears to be on the reactants side of reaction 2. This reductant-dependent shift in the kinetic and thermodynamic properties of the methyl group transfer reaction is not understood mechanistically. Bhaskar et al. have examined the steady-state Kinetics of the exchange reaction between acetyl-CoA and dephospho-CoA as catalyzed by the ACS/CODH homolog from Methanosarcina barkeri.22 Their results indicate a ping-pong mechanism in which the binding of acetyl-CoA to the enzyme is followed by the release of CoA and formation of the acetyl-intermediate. They proposed that acetyl-CoA binds to the oxidized form of the enzyme, followed by reduction. This was suggested because partially-reduced enzyme exhibited cooperative binding with acetyl-CoA whereas fully reduced enzyme showed simple hyperbolic binding. However, the same behavior would be observed if enzyme were first reduced and then bound with acetyl-CoA. This latter scenario would be congruent with a nucleophilic attack (e.g. by a Ni0 species) on the carbonyl of acetyl-CoA, as in the reverse of reaction 4. The alternative proposal of binding followed by reduction would seem to require attack by Ni2+, a non-nucleophilic metal ion. Using two methods, Bhaskar et al. measured the equilibrium constant for the reverse of reaction 4 to be ~ 0.2 (averaged value),22 suggesting KCoA ~ 5 for a homologous ACS/CODH from a methanogenic archaeon. To date, no direct studies of the CO insertion reaction 3 have been reported, nor have the Kinetics of the reductive elimination of the acetyl group and CoA, reaction 4, been reported. The problem in studying these reactions has been to identify strategies for monitoring them. Using Stopped-Flow Kinetics, we report here that reactions 3 and 4 can be monitored by starting with the methylated state of ACS/CODH. Resulting traces were used to construct a simple kinetic model describing the catalytic mechanism of acetyl-CoA synthase. In this paper we report these results and describe the model.
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function of the tunnel in acetylcoenzyme a synthase carbon monoxide dehydrogenase
Journal of Biological Inorganic Chemistry, 2006Co-Authors: Xiangshi Tan, Anne Volbeda, Juan C Fontecillacamps, Paul A LindahlAbstract:Acetylcoenzyme A synthase/carbon monoxide dehydrogenase (ACS/CODH) contains two Ni-Fe-S active-site clusters (called A and C) connected by a tunnel through which CO and CO2 migrate. Site-directed mutants A578C, L215F, and A219F were designed to block the tunnel at different points along the region between the two C-clusters. Two other mutant proteins F70W and N101Q were designed to block the region that connects the tunnel at the betabeta interface with a water channel also located at that interface. Purified mutant proteins were assayed for Ni/Fe content and examined by electron paramagnetic resonance spectroscopy. Analyses indicate that same metal clusters found in wild-type (WT) ACS/CODH (i.e., the A-, B-, C-, and probably D-clusters) are properly assembled in the mutant enzymes. Stopped-Flow Kinetics revealed that these centers in the mutants are rapidly reducible by dithionite but are only slowly reducible by CO, suggesting an impaired ability of CO to migrate through the tunnel to the C-cluster. Relative to the WT enzyme, mutant proteins exhibited little CODH or ACS activity (using CO2 as a substrate). Some ACS activity was observed when CO was a substrate, but not the cooperative CO inhibition effect characteristic of WT ACS/CODH. These results suggest that CO and CO2 enter and exit the enzyme at the water channel along the betabeta subunit interface. They also suggest two pathways for CO during synthesis of acetylcoenzyme A, including one in which CO enters the enzyme and migrates through the tunnel before binding at the A-cluster, and another in which CO binds the A-cluster directly from the solvent.
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stopped flow Kinetics of methyl group transfer between the corrinoid iron sulfur protein and acetyl coenzyme a synthase from clostridium thermoaceticum
Journal of the American Chemical Society, 2002Co-Authors: Xiangshi Tan, Christopher Sewell, Paul A LindahlAbstract:Kinetics of methyl group transfer between the Ni-Fe-S-containing acetyl-CoA synthase (ACS) and the corrinoid protein (CoFeSP) from Clostridium thermoaceticum were investigated using the Stopped-Flow method at 390 nm. Rates of the reaction CH(3)-Co(3+)FeSP + ACS(red) Co(1+)FeSP + CH(3)-ACS(ox) in both forward and reverse directions were determined using various protein and reductant concentrations. Ti(3+)citrate, dithionite, and CO were used to reductively activate ACS (forming ACS(red)). The simplest mechanism that adequately fit the data involved formation of a [CH(3)-Co(3+)FeSP]:[ACS(red)] complex, methyl group transfer (forming [Co(1+)FeSP]:[CH(3)-ACS(ox)]), product dissociation (forming Co(1+)FeSP + CH(3)-ACS(ox)), and CO binding yielding a nonproductive enzyme state (ACS(red) + CO ACS(red)-CO). Best-fit rate constants were obtained. CO inhibited methyl group transfer by binding ACS(red) in accordance with K(D) = 180 +/- 90 microM. Fits were unimproved when >1 CO was assumed to bind. Ti(3+)citrate and dithionite inhibited the reverse methyl group transfer reaction, probably by reducing the D-site of CH(3)-ACS(ox). This redox site is oxidized by 2e(-) when the methyl cation is transferred from CH(3)-Co(3+)FeSP to ACS(red), and is reduced during the reverse reaction. Best-fit K(D) values for pre- and post-methyl-transfer complexes were 0.12 +/- 0.06 and 0.3 +/- 0.2 microM, respectively. Intracomplex methyl group transfer was reversible with K(eq) = 2.3 +/- 0.9 (k(f)/k(r) = 6.9 s(-1)/3.0 s(-1)). The nucleophilicity of the [Ni(2+)D(red)] unit appears comparable to that of Co(1+) cobalamins. Reduction of the D-site may cause the Ni(2+) of the A-cluster to behave like the Ni of an organometallic Ni(0) complex.
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stopped flow Kinetics of methyl group transfer between the corrinoid iron sulfur protein and acetyl coenzyme a synthase from clostridium thermoaceticum
Journal of the American Chemical Society, 2002Co-Authors: Xiangshi Tan, Christopher Sewell, Paul A LindahlAbstract:Kinetics of methyl group transfer between the Ni−Fe−S-containing acetyl-CoA synthase (ACS) and the corrinoid protein (CoFeSP) from Clostridium thermoaceticum were investigated using the Stopped-Flow method at 390 nm. Rates of the reaction CH3-Co3+FeSP + ACSred ⇄ Co1+FeSP + CH3-ACSox in both forward and reverse directions were determined using various protein and reductant concentrations. Ti3+citrate, dithionite, and CO were used to reductively activate ACS (forming ACSred). The simplest mechanism that adequately fit the data involved formation of a [CH3-Co3+FeSP]:[ACSred] complex, methyl group transfer (forming [Co1+FeSP]:[CH3-ACSox]), product dissociation (forming Co1+FeSP + CH3-ACSox), and CO binding yielding a nonproductive enzyme state (ACSred + CO ⇄ ACSred-CO). Best-fit rate constants were obtained. CO inhibited methyl group transfer by binding ACSred in accordance with KD = 180 ± 90 μM. Fits were unimproved when >1 CO was assumed to bind. Ti3+citrate and dithionite inhibited the reverse methyl group...