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Karen S Anderson - One of the best experts on this subject based on the ideXlab platform.
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the kinetic mechanism of the human bifunctional enzyme atic 5 amino 4 imidazolecarboxamide ribonucleotide transformylase inosine 5 monophosphate cyclohydrolase a surprising lack of Substrate Channeling
Journal of Biological Chemistry, 2002Co-Authors: Karen G Bulock, Peter G Beardsley, Karen S AndersonAbstract:Abstract 5-Amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway forde novo synthesis of inosine 5′-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 ± 0.4 s−1) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s−1) is ∼2–3-fold faster than the forward rate (2.9 s−1), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of Substrate Channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.
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the kinetic mechanism of the human bifunctional enzyme atic 5 amino 4 imidazolecarboxamide ribonucleotide transformylase inosine 5 monophosphate cyclohydrolase a surprising lack of Substrate Channeling
Journal of Biological Chemistry, 2002Co-Authors: Karen G Bulock, Peter G Beardsley, Karen S AndersonAbstract:5-Amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway for de novo synthesis of inosine 5'-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 +/- 0.4 s(-1)) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s(-1)) is approximately 2-3-fold faster than the forward rate (2.9 s(-1)), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of Substrate Channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.
Karen G Bulock - One of the best experts on this subject based on the ideXlab platform.
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the kinetic mechanism of the human bifunctional enzyme atic 5 amino 4 imidazolecarboxamide ribonucleotide transformylase inosine 5 monophosphate cyclohydrolase a surprising lack of Substrate Channeling
Journal of Biological Chemistry, 2002Co-Authors: Karen G Bulock, Peter G Beardsley, Karen S AndersonAbstract:Abstract 5-Amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway forde novo synthesis of inosine 5′-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 ± 0.4 s−1) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s−1) is ∼2–3-fold faster than the forward rate (2.9 s−1), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of Substrate Channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.
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the kinetic mechanism of the human bifunctional enzyme atic 5 amino 4 imidazolecarboxamide ribonucleotide transformylase inosine 5 monophosphate cyclohydrolase a surprising lack of Substrate Channeling
Journal of Biological Chemistry, 2002Co-Authors: Karen G Bulock, Peter G Beardsley, Karen S AndersonAbstract:5-Amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway for de novo synthesis of inosine 5'-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 +/- 0.4 s(-1)) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s(-1)) is approximately 2-3-fold faster than the forward rate (2.9 s(-1)), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of Substrate Channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.
Michael F Dunn - One of the best experts on this subject based on the ideXlab platform.
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Allostery and Substrate Channeling in the Tryptophan Synthase Bienzyme Complex: Evidence for Two Subunit Conformations and Four Quaternary States
2016Co-Authors: Dimitri Niks, Adam T Dierkers, Dan Borchardt, Leonard J Mueller, Huu Ngo, Eduardo Hilario, Li Fan, Thomas J. Neubauer, Michael F DunnAbstract:The allosteric regulation of Substrate Channeling in tryptophan synthase involves ligand-mediated allosteric signaling that switches the α- and β-subunits between open (low activity) and closed (high activity) conformations. This switching prevents the escape of the common intermediate, indole, and synchronizes the α- and β-catalytic cycles. 19F NMR studies of bound α-site Substrate analogues, N-(4′-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4′-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), were found to be sensitive NMR probes of β-subunit conformation. Both the internal and external aldimine F6 complexes gave a single bound peak at the same chemical shift, while α-aminoacrylate and quinonoid F6 complexes all gave a different bound peak shifted by +1.07 ppm. The F9 complexes exhibited similar behavior, but with a corresponding shift of −0.12 ppm. X-ray crystal structures show the F6 and F9 CF3 groups located at the α–β subunit interface and report changes in both the ligand conformation and the surrounding protein microenvironment. Ab initio computational modeling suggests that the change in 19F chemical shift results primarily from changes in the α-site ligand conformation. Structures of α-aminoacrylate F6 and F9 complexes and quinonoid F6 and F9 complexes show the α- and β-subunits have closed conformations wherein access of ligands into the α- and β-sites from solution is blocked. Internal and external aldimine structures show the α- and β-subunits with closed and open global conformations, respectively. These results establish that β-subunits exist in two global conformational states, designated open, where the β-sites are freely accessible to Substrates, and closed, where the β-site portal into solution is blocked. Switching between these conformations is critically important for the αβ-catalytic cycle
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allostery and Substrate Channeling in the tryptophan synthase bienzyme complex evidence for two subunit conformations and four quaternary states
Biochemistry, 2013Co-Authors: Dimitri Niks, Adam T Dierkers, Dan Borchardt, Leonard J Mueller, Huu Ngo, Eduardo Hilario, Thomas J Neubauer, Li Fan, Michael F DunnAbstract:The allosteric regulation of Substrate Channeling in tryptophan synthase involves ligand-mediated allosteric signaling that switches the α- and β-subunits between open (low activity) and closed (high activity) conformations. This switching prevents the escape of the common intermediate, indole, and synchronizes the α- and β-catalytic cycles. 19F NMR studies of bound α-site Substrate analogues, N-(4′-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4′-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), were found to be sensitive NMR probes of β-subunit conformation. Both the internal and external aldimine F6 complexes gave a single bound peak at the same chemical shift, while α-aminoacrylate and quinonoid F6 complexes all gave a different bound peak shifted by +1.07 ppm. The F9 complexes exhibited similar behavior, but with a corresponding shift of −0.12 ppm. X-ray crystal structures show the F6 and F9 CF3 groups located at the α–β subunit interface and report changes in both the ligan...
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allosteric regulation of Substrate Channeling and catalysis in the tryptophan synthase bienzyme complex
Archives of Biochemistry and Biophysics, 2012Co-Authors: Michael F DunnAbstract:Abstract The tryptophan synthase α 2 β 2 bi-enzyme complex catalyzes the last two steps in the synthesis of l -tryptophan ( l -Trp). The α-subunit catalyzes cleavage of 3-indole- d -glycerol 3′-phosphate (IGP) to give indole and d -glyceraldehyde 3′-phosphate (G3P). Indole is then transferred (channeled) via an interconnecting 25 A-long tunnel, from the α-subunit to the β-subunit where it reacts with l -Ser in a pyridoxal 5′-phosphate-dependent reaction to give l -Trp and a water molecule. The efficient utilization of IGP and l -Ser by tryptophan synthase to synthesize l -Trp utilizes a system of allosteric interactions that (1) function to switch the α-site on and off at different stages of the β-subunit catalytic cycle, and (2) prevent the escape of the channeled intermediate, indole, from the confines of the α- and β-catalytic sites and the interconnecting tunnel. This review discusses in detail the chemical origins of the allosteric interactions responsible both for switching the α-site on and off, and for triggering the conformational changes between open and closed states which prevent the escape of indole from the bienzyme complex.
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tryptophan synthase structure and function of the monovalent cation site
Biochemistry, 2009Co-Authors: Adam T Dierkers, Dimitri Niks, I Schlichting, Michael F DunnAbstract:The monovalent cation (MVC) site of the tryptophan synthase from Salmonella typhimurium plays essential roles in catalysis and in the regulation of Substrate Channeling. In vitro, MVCs affect the equilibrium distribution of intermediates formed in the reaction of l-Ser with the α2β2 complex; the MVC-free, Cs+-bound, and NH4+-bound enzymes stabilize the α-aminoacrylate species, E(A-A), while Na+ binding stabilizes the l-Ser external aldimine species, E(Aex1). Two probes of β-site reactivity and conformation were used herein, the reactive indole analogue, indoline, and the l-Trp analogue, l-His. MVC-bound E(A-A) reacts rapidly with indoline to give the indoline quinonoid species, E(Q)indoline, which slowly converts to dihydroiso-l-tryptophan. MVC-free E(A-A) gives very little E(Q)indoline, and turnover is strongly impaired; the fraction of E(Q)indoline formed is <3.5% of that given by the Na+-bound form. The reaction of l-Ser with the MVC-free internal aldimine species, E(Ain), initially gives small amounts...
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Tryptophan synthase: the workings of a Channeling nanomachine.
Trends in biochemical sciences, 2008Co-Authors: Michael F Dunn, Thomas R M Barends, Dimitri Niks, Huu Ngo, Ilme SchlichtingAbstract:Substrate Channeling between enzymes has an important role in cellular metabolism by compartmentalizing cytoplasmic synthetic processes. The bacterial tryptophan synthases are multienzyme nanomachines that catalyze the last two steps in L-tryptophan biosynthesis. The common metabolite indole is transferred from one enzyme to the other in each αβ-dimeric unit of the α2β2 complex via an interconnecting 25-A-long tunnel. Recent solution studies of the Salmonella typhimurium α2β2 complex coupled with X-ray crystal-structure determinations of complexes with Substrates, intermediates and Substrate analogs have driven important breakthroughs concerning the identification of the linkages between the bi-enzyme complex structure, catalysis at the α- and β-active sites, and the allosteric regulation of Substrate Channeling.
Peter G Beardsley - One of the best experts on this subject based on the ideXlab platform.
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the kinetic mechanism of the human bifunctional enzyme atic 5 amino 4 imidazolecarboxamide ribonucleotide transformylase inosine 5 monophosphate cyclohydrolase a surprising lack of Substrate Channeling
Journal of Biological Chemistry, 2002Co-Authors: Karen G Bulock, Peter G Beardsley, Karen S AndersonAbstract:Abstract 5-Amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway forde novo synthesis of inosine 5′-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 ± 0.4 s−1) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s−1) is ∼2–3-fold faster than the forward rate (2.9 s−1), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of Substrate Channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.
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the kinetic mechanism of the human bifunctional enzyme atic 5 amino 4 imidazolecarboxamide ribonucleotide transformylase inosine 5 monophosphate cyclohydrolase a surprising lack of Substrate Channeling
Journal of Biological Chemistry, 2002Co-Authors: Karen G Bulock, Peter G Beardsley, Karen S AndersonAbstract:5-Amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway for de novo synthesis of inosine 5'-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 +/- 0.4 s(-1)) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s(-1)) is approximately 2-3-fold faster than the forward rate (2.9 s(-1)), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of Substrate Channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.
Shelley D. Minteer - One of the best experts on this subject based on the ideXlab platform.
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Substrate Channeling in an artificial metabolon a molecular dynamics blueprint for an experimental peptide bridge
ACS Catalysis, 2017Co-Authors: Yuanchao Liu, Shelley D. Minteer, David P Hickey, Jing Yao Guo, Erica Earl, Sofiene Abdellaoui, Ross D Milton, Matthew S Sigman, Scott Calabrese BartonAbstract:Natural enzyme cascades utilize electrostatic guidance as an effective technique to control the diffusion of charged reaction intermediates between catalytic active sites in a process known as Substrate Channeling. However, the limited understanding of Channeling mechanisms has abated the application of this technique in artificial catalytic cascades. In this work, we utilize molecular dynamics simulations to describe the transport of anionic intermediates (e.g., oxalate and glucose-6-phosphate) on a theoretical cationic α-helix peptide bridge and identify rules for molecular-level design of electrostatic Channeling. These simulations allowed us to elucidate a surface diffusion mechanism whereby the anionic intermediate undergoes discrete hydrogen-bonding interactions along adjacent cationic residues on the peptide bridge. Using MD simulations as a foundational blueprint, we synthesized an enzyme complex using a poly(lysine) peptide chain as a cationic bridge between glucose-6-phosphate dehydrogenase and ...
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Substrate Channeling in an Artificial Metabolon: A Molecular Dynamics Blueprint for an Experimental Peptide Bridge
2017Co-Authors: Yuanchao Liu, Shelley D. Minteer, David P Hickey, Jing Yao Guo, Erica Earl, Sofiene Abdellaoui, Ross D Milton, Matthew S Sigman, Scott Calabrese BartonAbstract:Natural enzyme cascades utilize electrostatic guidance as an effective technique to control the diffusion of charged reaction intermediates between catalytic active sites in a process known as Substrate Channeling. However, the limited understanding of Channeling mechanisms has abated the application of this technique in artificial catalytic cascades. In this work, we utilize molecular dynamics simulations to describe the transport of anionic intermediates (e.g., oxalate and glucose-6-phosphate) on a theoretical cationic α-helix peptide bridge and identify rules for molecular-level design of electrostatic Channeling. These simulations allowed us to elucidate a surface diffusion mechanism whereby the anionic intermediate undergoes discrete hydrogen-bonding interactions along adjacent cationic residues on the peptide bridge. Using MD simulations as a foundational blueprint, we synthesized an enzyme complex using a poly(lysine) peptide chain as a cationic bridge between glucose-6-phosphate dehydrogenase and hexokinase. Stopped-flow lag time experiments demonstrate the ability of the artificially linked enzyme complex to facilitate electrostatic Substrate Channeling, while an analogous neutral poly(glycine)-bridged complex was used as a control to isolate proximity effects from artificial Substrate Channeling
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Improving the Performance of Methanol Biofuel Cells Utilizing an Enzyme Cascade Bioanode with DNA-Bridged Substrate Channeling
2017Co-Authors: Lin Xia, Scott Banta, Khiem Van Nguyen, Yaovi Holade, Han Han, Kevin Dooley, Plamen Atanassov, Shelley D. MinteerAbstract:The development of enzymatic biofuel cells has been plagued by the high cost of enzyme purification and low efficiency of fuel oxidation. Here, we demonstrate a protein purification-free approach to assemble an alcohol dehydrogenase and aldehyde dehydrogenase enzyme cascade-based bioanode for use in a methanol biofuel cell. Each enzyme was fused to a different sequence-specific zinc finger DNA-binding protein. The zinc finger domains serve as both tags to isolate the enzymes from crude cell lysates as well as anchors to immobilize the enzymes on DNA-modified multiwalled carbon nanotubes. The biofuel cells based on the enzyme cascade bioanodes show a maximum power output of 24.5 ± 3.2 μW cm–2, which is comparable to fuel cells utilizing purified enzymes. Further analysis of kinetic behavior revealed a significant increase in the reactivity of the complexes due to Substrate Channeling of the aldehyde intermediate
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direct evidence for metabolon formation and Substrate Channeling in recombinant tca cycle enzymes
ACS Chemical Biology, 2016Co-Authors: Beyza Bulutoglu, Shelley D. Minteer, Kristen E Garcia, Scott BantaAbstract:Supramolecular assembly of enzymes into metabolon structures is thought to enable efficient transport of reactants between active sites via Substrate Channeling. Recombinant versions of porcine citrate synthase (CS), mitochondrial malate dehydrogenase (mMDH), and aconitase (Aco) were found to adopt a homogeneous native-like metabolon structure in vitro. Site-directed mutagenesis performed on highly conserved arginine residues located in the positively charged channel connecting mMDH and CS active sites led to the identification of CS(R65A) which retained high catalytic efficiency. Substrate Channeling between the CS mutant and mMDH was severely impaired and the overall Channeling probability decreased from 0.99 to 0.023. This work provides direct mechanistic evidence for the Channeling of reaction intermediates, and disruption of this interaction would have important implications on the control of flux in central carbon metabolism.
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Direct Evidence for Metabolon Formation and Substrate Channeling in Recombinant TCA Cycle Enzymes
2016Co-Authors: Beyza Bulutoglu, Shelley D. Minteer, Kristen E Garcia, Scott BantaAbstract:Supramolecular assembly of enzymes into metabolon structures is thought to enable efficient transport of reactants between active sites via Substrate Channeling. Recombinant versions of porcine citrate synthase (CS), mitochondrial malate dehydrogenase (mMDH), and aconitase (Aco) were found to adopt a homogeneous native-like metabolon structure in vitro. Site-directed mutagenesis performed on highly conserved arginine residues located in the positively charged channel connecting mMDH and CS active sites led to the identification of CS(R65A) which retained high catalytic efficiency. Substrate Channeling between the CS mutant and mMDH was severely impaired and the overall Channeling probability decreased from 0.99 to 0.023. This work provides direct mechanistic evidence for the Channeling of reaction intermediates, and disruption of this interaction would have important implications on the control of flux in central carbon metabolism
