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Papers by n.ravi Kumar
Biochemistry, 1995
We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-C... more We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-CoA dehydrogenase (HMCAD). This procedure routinely yields 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 Cg-CoA molecules (viz., octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation at the a-P carbon centers; 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 Cg-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 Cg-CoA molecules. However, the electronic structural changes accompanying the 2-octynoyl-CoA-dependent inactivation of the 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 Cg-CoA binding site. In a series of publications over the past four years, we have elaborated on several mechanistic aspects of the medium-chain acyl-CoA dehydrogenase (MCAD)-cataly zed' "dehydrogenase" and "oxidase" reactions (
Biochemistry, 1995
In a previous paper, we demonstrated that the reductive half-reaction of medium-chain fatty acyl-... more In a previous paper, we demonstrated that the reductive half-reaction of medium-chain fatty acyl-CoA dehydrogenase (MCAD), utilizing octanoyl-CoA as physiological substrate, generates two (kinetically distinct) forms of the reduced enzyme (MCAD-FADH2) - octenoyl-CoA charge-transfer complexes [Kumar, N.R., & Srivastava, D.K. (1994) Biochemistry 33, 8833-8841]. We present evidence that octenoyl-CoA dissociates from the second (most stable) charge-transfer complex (referred to as CT2) via two alternative ("facile" and "restricted") pathways. The dissociation of octenoyl-CoA via the facile pathway involves the reversal of the overall reductive half-reaction of the enzyme, generating MCAD-FAD - octanoyl-CoA as the Michaelis complex, followed by dissociation of the latter complex into MCAD-FAD + octanoyl-CoA. Hence, via this pathway, octenoyl-CoA is released from the enzyme site in the form of octanoyl-CoA. In contrast, the restricted pathway involves a direct (albeit slow) dissociation of octenoyl-CoA from CT2 to yield MCAD-FADH2 + octenoyl-CoA. The kinetic profile for the dissociation of octenoyl-CoA via the restricted pathway matches the rate of oxidation of the reduced flavin (within CT2) by O2. This suggests that the oxidase activity of the enzyme remains suppressed as long as the reduced enzyme predominates in the form of the charge-transfer complex(es). The oxidase activity of the enzyme emerges concomitantly with the conversion of CT2 to the MCAD-FADH2 - octenoyl-CoA Michaelis complex. The energetic basis for the dissociation of octenoyl-CoA via the facile and restricted pathways and the mechanism of suppression of the oxidase activity of the enzyme are discussed.
Analytical Biochemistry, 1992
We offer a new titration protocol for determining the dissociation constant and binding stoichiom... more We offer a new titration protocol for determining the dissociation constant and binding stoichiometry of protein-ligand complex, detectable by spectroscopic methods. This approach neither is limited to the range of protein or ligand concentrations employed during titration experiment nor relies on precise determinations of the titration "endpoint," i.e., the maximal signal changes upon saturation of protein by ligand (or vice versa). In this procedure, a fixed concentration of protein (or ligand) is titrated by increasing volumes of a stock ligand (or protein) solution, and the changes in the spectroscopic signal are recorded after each addition of the titrant. The signal for interaction between protein and ligand first increases, reaches a maximum value, and then starts decreasing due to dilution effect. The volume of the titrant required to achieve the maximum signal changes is utilized to calculate the dissociation constant and the binding stoichiometry of the protein-ligand complex according to the theoretical relationships developed herein. This procedure has been tested for the interaction of avidin with a chromophoric biotin analogue, 2-(4'-hydroxyazobenzene)benzoic acid by following the absorption signal of their interaction at 500 nm. The widespread applicability of this procedure to protein-ligand complexes detected by other spectroscopic techniques and its advantages over conventional methods are discussed.
Acta Crystallographica Section E Structure Reports Online, 2011
Biochemistry, 1995
We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-C... more We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-CoA dehydrogenase (HMCAD). This procedure routinely yields 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 Cg-CoA molecules (viz., octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation at the a-P carbon centers; 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 Cg-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 Cg-CoA molecules. However, the electronic structural changes accompanying the 2-octynoyl-CoA-dependent inactivation of the 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 Cg-CoA binding site. In a series of publications over the past four years, we have elaborated on several mechanistic aspects of the medium-chain acyl-CoA dehydrogenase (MCAD)-cataly zed' "dehydrogenase" and "oxidase" reactions (
Biochemistry, 1995
In a previous paper, we demonstrated that the reductive half-reaction of medium-chain fatty acyl-... more In a previous paper, we demonstrated that the reductive half-reaction of medium-chain fatty acyl-CoA dehydrogenase (MCAD), utilizing octanoyl-CoA as physiological substrate, generates two (kinetically distinct) forms of the reduced enzyme (MCAD-FADH2) - octenoyl-CoA charge-transfer complexes [Kumar, N.R., & Srivastava, D.K. (1994) Biochemistry 33, 8833-8841]. We present evidence that octenoyl-CoA dissociates from the second (most stable) charge-transfer complex (referred to as CT2) via two alternative ("facile" and "restricted") pathways. The dissociation of octenoyl-CoA via the facile pathway involves the reversal of the overall reductive half-reaction of the enzyme, generating MCAD-FAD - octanoyl-CoA as the Michaelis complex, followed by dissociation of the latter complex into MCAD-FAD + octanoyl-CoA. Hence, via this pathway, octenoyl-CoA is released from the enzyme site in the form of octanoyl-CoA. In contrast, the restricted pathway involves a direct (albeit slow) dissociation of octenoyl-CoA from CT2 to yield MCAD-FADH2 + octenoyl-CoA. The kinetic profile for the dissociation of octenoyl-CoA via the restricted pathway matches the rate of oxidation of the reduced flavin (within CT2) by O2. This suggests that the oxidase activity of the enzyme remains suppressed as long as the reduced enzyme predominates in the form of the charge-transfer complex(es). The oxidase activity of the enzyme emerges concomitantly with the conversion of CT2 to the MCAD-FADH2 - octenoyl-CoA Michaelis complex. The energetic basis for the dissociation of octenoyl-CoA via the facile and restricted pathways and the mechanism of suppression of the oxidase activity of the enzyme are discussed.
Analytical Biochemistry, 1992
We offer a new titration protocol for determining the dissociation constant and binding stoichiom... more We offer a new titration protocol for determining the dissociation constant and binding stoichiometry of protein-ligand complex, detectable by spectroscopic methods. This approach neither is limited to the range of protein or ligand concentrations employed during titration experiment nor relies on precise determinations of the titration "endpoint," i.e., the maximal signal changes upon saturation of protein by ligand (or vice versa). In this procedure, a fixed concentration of protein (or ligand) is titrated by increasing volumes of a stock ligand (or protein) solution, and the changes in the spectroscopic signal are recorded after each addition of the titrant. The signal for interaction between protein and ligand first increases, reaches a maximum value, and then starts decreasing due to dilution effect. The volume of the titrant required to achieve the maximum signal changes is utilized to calculate the dissociation constant and the binding stoichiometry of the protein-ligand complex according to the theoretical relationships developed herein. This procedure has been tested for the interaction of avidin with a chromophoric biotin analogue, 2-(4'-hydroxyazobenzene)benzoic acid by following the absorption signal of their interaction at 500 nm. The widespread applicability of this procedure to protein-ligand complexes detected by other spectroscopic techniques and its advantages over conventional methods are discussed.
Acta Crystallographica Section E Structure Reports Online, 2011