The Experts below are selected from a list of 198 Experts worldwide ranked by ideXlab platform
Rikkert K Wierenga - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of human mitochondrial 3 ketoacyl coa thiolase t1 insight into the reaction mechanism of its thiolase and thioesterase activities
Acta Crystallographica Section D-biological Crystallography, 2014Co-Authors: Tiila-riikka Kiema, Rajesh K Harijan, Malgorzata Strozyk, Stefan E. H. Alexson, Toshiyuki Fukao, Rikkert K WierengaAbstract:Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 A resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with Octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.
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The crystal structure of human mitochondrial 3‐ketoacyl‐CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities
Acta Crystallographica Section D-biological Crystallography, 2014Co-Authors: Tiila-riikka Kiema, Rajesh K Harijan, Malgorzata Strozyk, Stefan E. H. Alexson, Toshiyuki Fukao, Rikkert K WierengaAbstract:Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 A resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with Octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.
Tiila-riikka Kiema - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of human mitochondrial 3 ketoacyl coa thiolase t1 insight into the reaction mechanism of its thiolase and thioesterase activities
Acta Crystallographica Section D-biological Crystallography, 2014Co-Authors: Tiila-riikka Kiema, Rajesh K Harijan, Malgorzata Strozyk, Stefan E. H. Alexson, Toshiyuki Fukao, Rikkert K WierengaAbstract:Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 A resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with Octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.
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The crystal structure of human mitochondrial 3‐ketoacyl‐CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities
Acta Crystallographica Section D-biological Crystallography, 2014Co-Authors: Tiila-riikka Kiema, Rajesh K Harijan, Malgorzata Strozyk, Stefan E. H. Alexson, Toshiyuki Fukao, Rikkert K WierengaAbstract:Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 A resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with Octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.
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The crystal structure of enoyl-CoA hydratase complexed with Octanoyl-CoA reveals the structural adaptations required for binding of a long chain fatty acid-CoA molecule.
Journal of molecular biology, 1998Co-Authors: C. K. Engel, Tiila-riikka Kiema, J.k. Hiltunen, R.k. WierengaAbstract:The structure of the hexameric rat mitochondrial enoyl-Coenzyme A (CoA) hydratase, co-crystallised with the inhibitor Octanoyl-CoA, has been refined at a resolution of 2.4 A. Enoyl-CoA hydratase catalyses the hydration of 2,3-unsaturated enoyl-CoA thioesters. In the crystal structure only four of the six active sites of the hexamer in the asymmetric unit are occupied with a ligand molecule, showing an unliganded and a liganded active site within the same crystal form. While the protein assembly and fold is identical to the previously solved acetoacetyl-CoA complex, differences are observed close to the fatty acid binding pocket due to the different nature of the ligands. The fatty acid tail of Octanoyl-CoA is bound in an extended conformation. This is possible because a high B-factor loop, which separates in the acetoacetyl-CoA complex the binding pocket of the acetoacetyl-CoA fatty acid tail from the intertrimer space, has moved aside to allow binding of the longer Octanoyl-CoA moiety. The movement of this loop opens a tunnel which traverses the complete subunit from the solvent space to the intertrimer space. The conformation of the catalytic residues is identical, in both structures as well as in the liganded and the unliganded active sites. In the unliganded active sites a water molecules is bound between the two catalytic glutamate, residues Glu144 and Glu164. After superposition of a liganded active site on an unliganded active site this water molecule is close to the carbon centre that becomes hydroxylated in the hydratase reaction. These findings support the idea that the active site is rigid and that the catalytic residues and the water molecule, as seen in the unliganded active site, are pre-positioned for very efficient catalysis.
Eckhard Boles - One of the best experts on this subject based on the ideXlab platform.
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An engineered fatty acid synthase combined with a carboxylic acid reductase enables de novo production of 1-octanol in Saccharomyces cerevisiae
Biotechnology for Biofuels, 2018Co-Authors: Sandra Henritzi, Martin Grininger, Manuel Fischer, Mislav Oreb, Eckhard BolesAbstract:BackgroundThe ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase-mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway.ResultsThe previously engineered short-chain acyl-CoA producing yeast Fas1R1834K/Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involvement of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized Octanoyl-CoA was converted to 1-octanol up to a titer of 26.0 mg L−1 in a 72-h fermentation. The additional accumulation of 90 mg L−1 octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was supplied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escherichia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L−1. However, in growth tests concentrations even lower than 50.0 mg L−1 turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to the organic phase, preventing its re-uptake.ConclusionsBy providing chain length control via an engineered Octanoyl-CoA producing fatty acid synthase, we were able to specifically produce 1-octanol with S. cerevisiae. Before metabolic engineering can be used to further increase product titers and yields, strategies must be developed that cope with the toxic effects of 1-octanol on the yeast cells.
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An engineered fatty acid synthase combined with a carboxylic acid reductase enables de novo production of 1-octanol in Saccharomyces cerevisiae.
Biotechnology for biofuels, 2018Co-Authors: Sandra Henritzi, Martin Grininger, Manuel Fischer, Mislav Oreb, Eckhard BolesAbstract:The ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase-mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway. The previously engineered short-chain acyl-CoA producing yeast Fas1R1834K/Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involvement of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized Octanoyl-CoA was converted to 1-octanol up to a titer of 26.0 mg L-1 in a 72-h fermentation. The additional accumulation of 90 mg L-1 octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was supplied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escherichia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L-1. However, in growth tests concentrations even lower than 50.0 mg L-1 turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to the organic phase, preventing its re-uptake. By providing chain length control via an engineered Octanoyl-CoA producing fatty acid synthase, we were able to specifically produce 1-octanol with S. cerevisiae. Before metabolic engineering can be used to further increase product titers and yields, strategies must be developed that cope with the toxic effects of 1-octanol on the yeast cells.
Dinesh Srivastava - One of the best experts on this subject based on the ideXlab platform.
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Thermodynamics of Ligand Binding and Catalysis in Human Liver Medium-Chain Acyl-CoA Dehydrogenase: Comparative Studies Involving Normal and 3‘-Dephosphorylated C8-CoAs and Wild-Type and Asn191 → Ala (N191A) Mutant Enzymes†
Biochemistry, 1998Co-Authors: Kevin L. Peterson, K. M. Peterson, Dinesh SrivastavaAbstract:Following our demonstration that the terminal 3‘-phosphate group of acyl-CoA substrates (which is confined to the exterior of the protein structure, and is fully exposed to the outside solvent environment) exhibits a functional role in the recombinant human liver medium-chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction [Peterson, K. L., and Srivastava, D. K. (1997) Biochem. J. 325, 751−760], we became interested in delineating its thermodynamic contribution in stabilizing the “ground” and “transition” state structures during enzyme catalysis. Since the 3‘-phosphate group of the coenzyme A thiolester has the potential to form a hydrogen bond with the side chain group of Asn-191, these studies were performed utilizing both normal and 3‘-dephosphorylated forms of Octanoyl-CoA and octenoyl-CoA (cumulatively referred to as C8-CoA) as the physiological substrate and product of the enzyme, respectively, as well as utilizing wild-type and Asn191 → Ala (N191A) site-specific mutant enzymes. The experimental da...
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Influence of excision of a methylene group from Glu-376 (Glu376-->Asp mutation) in the medium chain acyl-CoA dehydrogenase-catalyzed reaction.
Biochemistry, 1998Co-Authors: Kevin L. Peterson, David S. Galitz, Dinesh SrivastavaAbstract:The human liver medium chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction proceeds via abstraction of an alpha-proton from the acyl-CoA substrates by the carboxyl group of Glu-376. By using the methods of site-directed mutagenesis, we replaced Glu-376 by Asp (E376D mutation), expressed the wild-type and mutant enzymes in Escherichia coli, purified them to homogeneity, and compared their kinetic properties. The steady-state kinetic data revealed that the E376D mutation impaired (by about 15-20-fold) the turnover rate of the enzyme as well as its inactivation by 2-octynoyl-CoA. There was no selective solvent deuterium isotope effect on enzyme catalysis. These results lead to the suggestion that the carboxyl group of Asp-376 does not serve as efficient catalytic base as the carboxyl group of Glu-376. The E376D mutation impaired the Octanoyl-CoA-dependent reductive half-reaction such that the rate-limiting step of enzyme catalysis shifted from the product dissociation step (in the case of the wild-type enzyme) to the flavin reduction step, and abolished the previously noted kinetic and thermodynamic correspondences between the Octanoyl-CoA-dependent reductive half-reaction and the enzyme-octenoyl-CoA interaction [Kumar, N. R., and Srivastava, D. K. (1994) Biochemistry 33, 8833-8841]. Arguments are presented that the Glu-376-->Asp mutation results in uncoupling between the proton transfer and protein conformational change steps during enzyme catalysis.
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Recombinant human liver medium-chain acyl-CoA dehydrogenase: purification, characterization, and the mechanism of interactions with functionally diverse C8-CoA molecules.
Biochemistry, 1995Co-Authors: Kevin L. Peterson, Wu Y, John B. Shabb, Dinesh SrivastavaAbstract:We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-CoA dehydrogenase (HMCAD). This procedure routinely yield 100-150 mg of homogeneous preparation of the enzyme from 80 L of the Escherichia coli host cells. A comparative investigation of kinetic properties of the human liver and pig kidney enzymes revealed that, except for a few minor differences, both of these enzymes are nearly identical. We undertook detailed kinetic and thermodynamic investigations for the interaction of HMCAD-FAD with three C8-CoA molecules (viz., Octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation of the alpha-beta carbon center; Octanoyl-CoA and 2-octenoyl-CoA serve as the substrate and product of the enzyme, respectively, whereas 2-octynoyl-CoA is known to inactivate the enzyme. Our experimental results demonstrate that all three C8-CoA molecules first interact with HMCAD-FAD to form corresponding Michaelis complexes, followed by two subsequent isomerization reactions. The latter accompany either subtle changes in the electronic structures of the individual components (in case of 2-octenoyl-CoA and 2-octynoyl-CoA ligands), or a near-complete reduction of the enzyme-bound flavin (in case of Octanoyl-CoA). The rate and equilibrium constants intrinsic to the above microscopic steps exhibit marked similarity with different C8-CoA molecules. However, the electronic structural changes accompanying the 2-octynoyl-CoA-dependent inactivation of enzyme is 3-4 orders of magnitude slower than the above isomerization reactions. Hence, the Octanoyl-CoA-dependent reductive half-reaction and the 2-octynoyl-CoA-dependent covalent modification of the enzyme occur during entirely different microscopic steps. Arguments are presented that the origin of the above difference lies in the protein conformation-dependent orientation of Glu-376 in the vicinity of the C8-CoA binding site.
Kevin L. Peterson - One of the best experts on this subject based on the ideXlab platform.
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Thermodynamics of Ligand Binding and Catalysis in Human Liver Medium-Chain Acyl-CoA Dehydrogenase: Comparative Studies Involving Normal and 3‘-Dephosphorylated C8-CoAs and Wild-Type and Asn191 → Ala (N191A) Mutant Enzymes†
Biochemistry, 1998Co-Authors: Kevin L. Peterson, K. M. Peterson, Dinesh SrivastavaAbstract:Following our demonstration that the terminal 3‘-phosphate group of acyl-CoA substrates (which is confined to the exterior of the protein structure, and is fully exposed to the outside solvent environment) exhibits a functional role in the recombinant human liver medium-chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction [Peterson, K. L., and Srivastava, D. K. (1997) Biochem. J. 325, 751−760], we became interested in delineating its thermodynamic contribution in stabilizing the “ground” and “transition” state structures during enzyme catalysis. Since the 3‘-phosphate group of the coenzyme A thiolester has the potential to form a hydrogen bond with the side chain group of Asn-191, these studies were performed utilizing both normal and 3‘-dephosphorylated forms of Octanoyl-CoA and octenoyl-CoA (cumulatively referred to as C8-CoA) as the physiological substrate and product of the enzyme, respectively, as well as utilizing wild-type and Asn191 → Ala (N191A) site-specific mutant enzymes. The experimental da...
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Influence of excision of a methylene group from Glu-376 (Glu376-->Asp mutation) in the medium chain acyl-CoA dehydrogenase-catalyzed reaction.
Biochemistry, 1998Co-Authors: Kevin L. Peterson, David S. Galitz, Dinesh SrivastavaAbstract:The human liver medium chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction proceeds via abstraction of an alpha-proton from the acyl-CoA substrates by the carboxyl group of Glu-376. By using the methods of site-directed mutagenesis, we replaced Glu-376 by Asp (E376D mutation), expressed the wild-type and mutant enzymes in Escherichia coli, purified them to homogeneity, and compared their kinetic properties. The steady-state kinetic data revealed that the E376D mutation impaired (by about 15-20-fold) the turnover rate of the enzyme as well as its inactivation by 2-octynoyl-CoA. There was no selective solvent deuterium isotope effect on enzyme catalysis. These results lead to the suggestion that the carboxyl group of Asp-376 does not serve as efficient catalytic base as the carboxyl group of Glu-376. The E376D mutation impaired the Octanoyl-CoA-dependent reductive half-reaction such that the rate-limiting step of enzyme catalysis shifted from the product dissociation step (in the case of the wild-type enzyme) to the flavin reduction step, and abolished the previously noted kinetic and thermodynamic correspondences between the Octanoyl-CoA-dependent reductive half-reaction and the enzyme-octenoyl-CoA interaction [Kumar, N. R., and Srivastava, D. K. (1994) Biochemistry 33, 8833-8841]. Arguments are presented that the Glu-376-->Asp mutation results in uncoupling between the proton transfer and protein conformational change steps during enzyme catalysis.
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Functional role of a distal (3'-phosphate) group of CoA in the recombinant human liver medium-chain acyl-CoA dehydrogenase-catalysed reaction.
Biochemical Journal, 1997Co-Authors: Kevin L. Peterson, D K SrivastavaAbstract:The X-ray crystallographic structure of medium-chain acyl-CoA dehydrogenase (MCAD)–octenoyl-CoA complex reveals that the 3′-phosphate group of CoA is confined to the exterior of the protein structure [approx. 15 A (1.5 nm) away from the enzyme active site], and is fully exposed to the outside solvent environment. To ascertain whether such a distal (3′-phosphate) fragment of CoA plays any significant role in the enzyme catalysis, we investigated the recombinant human liver MCAD (HMCAD)-catalysed reaction by using normal (phospho) and 3′-phosphate-truncated (dephospho) forms of Octanoyl-CoA and butyryl-CoA substrates. The steady-state kinetic data revealed that deletion of the 3′-phosphate group from Octanoyl-CoA substrate increased the turnover rate of the enzyme to about one-quarter, whereas that from butyryl-CoA substrate decreased the turnover rate of the enzyme to about one-fifth; the K m values of both these substrates were increased by 5–10-fold on deletion of the 3′-phosphate group from the corresponding acyl-CoA substrates. The transient kinetics for the reductive half-reaction, oxidative half-reaction and the dissociation ‘off-rate’ (of the reaction product from the oxidized enzyme site) were all found to be affected by deletions of the 3′-phosphate group from Octanoyl-CoA and butyryl-CoA substrates. A cumulative account of these results reveals that, although the 3′-phosphate group of acyl-CoA substrates might seem ‘useless’ on the basis of the structural data, it has an essential functional role during HMCAD catalysis.
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Recombinant human liver medium-chain acyl-CoA dehydrogenase: purification, characterization, and the mechanism of interactions with functionally diverse C8-CoA molecules.
Biochemistry, 1995Co-Authors: Kevin L. Peterson, Wu Y, John B. Shabb, Dinesh SrivastavaAbstract:We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-CoA dehydrogenase (HMCAD). This procedure routinely yield 100-150 mg of homogeneous preparation of the enzyme from 80 L of the Escherichia coli host cells. A comparative investigation of kinetic properties of the human liver and pig kidney enzymes revealed that, except for a few minor differences, both of these enzymes are nearly identical. We undertook detailed kinetic and thermodynamic investigations for the interaction of HMCAD-FAD with three C8-CoA molecules (viz., Octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation of the alpha-beta carbon center; Octanoyl-CoA and 2-octenoyl-CoA serve as the substrate and product of the enzyme, respectively, whereas 2-octynoyl-CoA is known to inactivate the enzyme. Our experimental results demonstrate that all three C8-CoA molecules first interact with HMCAD-FAD to form corresponding Michaelis complexes, followed by two subsequent isomerization reactions. The latter accompany either subtle changes in the electronic structures of the individual components (in case of 2-octenoyl-CoA and 2-octynoyl-CoA ligands), or a near-complete reduction of the enzyme-bound flavin (in case of Octanoyl-CoA). The rate and equilibrium constants intrinsic to the above microscopic steps exhibit marked similarity with different C8-CoA molecules. However, the electronic structural changes accompanying the 2-octynoyl-CoA-dependent inactivation of enzyme is 3-4 orders of magnitude slower than the above isomerization reactions. Hence, the Octanoyl-CoA-dependent reductive half-reaction and the 2-octynoyl-CoA-dependent covalent modification of the enzyme occur during entirely different microscopic steps. Arguments are presented that the origin of the above difference lies in the protein conformation-dependent orientation of Glu-376 in the vicinity of the C8-CoA binding site.
