An approach for the determination of equilibrium constant of structural motility. Characterization of the fluctuational motion around residue Cys 153 of D glyceraldehyde 3 phosphate dehydrogenase

M. Vas, L. Boross

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Abstract

A kinetic approach is presented for the determination of equilibrium constant of a local conformational fluctuation of a protein molecule. As a result of this fluctuation a distinct side chain becomes temporarily accessible to a specific reagent. Determination of fluctuation constant, K(f) is based on the kinetic analysis of chemical modification of the side chain with a selective reagent which reacts faster than the conformational changes occur. The use of p mercuribenzoate mercaptide of 2 nitro 5 mercaptobenzoate as an -SH reagent is described to determine K(f) of D glyceralgehyde 3 phosphate dehydrogenase around residue Cys 153, when the hyperreactive side chain Cys 149 was previously carboxymethylated (CM enzyme). The values of K(f) are 0.034 ± 0.005 and 0.014 ± 0.003 in the case of CM apoenzyme and holoemzyme, respectively, in Tris HCl buffer, I = 0.05, pH 7.5, at 5°C. The free enthalpy change (ΔG) accompanying the conformational change which makes Cys 153 accessible to the reagent, is 1.86 ± 0.08 and 2.34 ± 0.11 kcal/mol for CM apoenzyme and holoenzyme, respectively. The enthalpy change (ΔH) is + 3.9 ± 0.7 kcal/mol both in the absence and presence of coenzyme. The entropy change (ΔS) is + 7.6 ± 1.3 and + 6.3 ± 1.1 calXdegree-1Xmol-1 for CM apoenzyme and holoenzyme, respectively. Based on the value of K(f) and the earlier kinetic data for mercaptide formation of Cys 153 one can characterize the structural motility: the rate constants of exposure and masking of residue Cys 153 of the apoenzyme are K(+1) = 2.2 ± 0.2 min-1 and K(-1) = 65 ± 16 min-1 respectively, under the experimental conditions.

Original languageEnglish
Pages (from-to)237-244
Number of pages8
JournalEuropean Journal of Biochemistry
Volume43
Issue number2
Publication statusPublished - 1974

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Apoenzymes
Glyceraldehyde-3-Phosphate Dehydrogenases
Equilibrium constants
Holoenzymes
Kinetics
Enthalpy
Sulfhydryl Reagents
Tromethamine
Coenzymes
Chemical modification
Entropy
Rate constants
Buffers
Oxidoreductases
Phosphates
Molecules
Enzymes
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "An approach for the determination of equilibrium constant of structural motility. Characterization of the fluctuational motion around residue Cys 153 of D glyceraldehyde 3 phosphate dehydrogenase",
abstract = "A kinetic approach is presented for the determination of equilibrium constant of a local conformational fluctuation of a protein molecule. As a result of this fluctuation a distinct side chain becomes temporarily accessible to a specific reagent. Determination of fluctuation constant, K(f) is based on the kinetic analysis of chemical modification of the side chain with a selective reagent which reacts faster than the conformational changes occur. The use of p mercuribenzoate mercaptide of 2 nitro 5 mercaptobenzoate as an -SH reagent is described to determine K(f) of D glyceralgehyde 3 phosphate dehydrogenase around residue Cys 153, when the hyperreactive side chain Cys 149 was previously carboxymethylated (CM enzyme). The values of K(f) are 0.034 ± 0.005 and 0.014 ± 0.003 in the case of CM apoenzyme and holoemzyme, respectively, in Tris HCl buffer, I = 0.05, pH 7.5, at 5°C. The free enthalpy change (ΔG) accompanying the conformational change which makes Cys 153 accessible to the reagent, is 1.86 ± 0.08 and 2.34 ± 0.11 kcal/mol for CM apoenzyme and holoenzyme, respectively. The enthalpy change (ΔH) is + 3.9 ± 0.7 kcal/mol both in the absence and presence of coenzyme. The entropy change (ΔS) is + 7.6 ± 1.3 and + 6.3 ± 1.1 calXdegree-1Xmol-1 for CM apoenzyme and holoenzyme, respectively. Based on the value of K(f) and the earlier kinetic data for mercaptide formation of Cys 153 one can characterize the structural motility: the rate constants of exposure and masking of residue Cys 153 of the apoenzyme are K(+1) = 2.2 ± 0.2 min-1 and K(-1) = 65 ± 16 min-1 respectively, under the experimental conditions.",
author = "M. Vas and L. Boross",
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T1 - An approach for the determination of equilibrium constant of structural motility. Characterization of the fluctuational motion around residue Cys 153 of D glyceraldehyde 3 phosphate dehydrogenase

AU - Vas, M.

AU - Boross, L.

PY - 1974

Y1 - 1974

N2 - A kinetic approach is presented for the determination of equilibrium constant of a local conformational fluctuation of a protein molecule. As a result of this fluctuation a distinct side chain becomes temporarily accessible to a specific reagent. Determination of fluctuation constant, K(f) is based on the kinetic analysis of chemical modification of the side chain with a selective reagent which reacts faster than the conformational changes occur. The use of p mercuribenzoate mercaptide of 2 nitro 5 mercaptobenzoate as an -SH reagent is described to determine K(f) of D glyceralgehyde 3 phosphate dehydrogenase around residue Cys 153, when the hyperreactive side chain Cys 149 was previously carboxymethylated (CM enzyme). The values of K(f) are 0.034 ± 0.005 and 0.014 ± 0.003 in the case of CM apoenzyme and holoemzyme, respectively, in Tris HCl buffer, I = 0.05, pH 7.5, at 5°C. The free enthalpy change (ΔG) accompanying the conformational change which makes Cys 153 accessible to the reagent, is 1.86 ± 0.08 and 2.34 ± 0.11 kcal/mol for CM apoenzyme and holoenzyme, respectively. The enthalpy change (ΔH) is + 3.9 ± 0.7 kcal/mol both in the absence and presence of coenzyme. The entropy change (ΔS) is + 7.6 ± 1.3 and + 6.3 ± 1.1 calXdegree-1Xmol-1 for CM apoenzyme and holoenzyme, respectively. Based on the value of K(f) and the earlier kinetic data for mercaptide formation of Cys 153 one can characterize the structural motility: the rate constants of exposure and masking of residue Cys 153 of the apoenzyme are K(+1) = 2.2 ± 0.2 min-1 and K(-1) = 65 ± 16 min-1 respectively, under the experimental conditions.

AB - A kinetic approach is presented for the determination of equilibrium constant of a local conformational fluctuation of a protein molecule. As a result of this fluctuation a distinct side chain becomes temporarily accessible to a specific reagent. Determination of fluctuation constant, K(f) is based on the kinetic analysis of chemical modification of the side chain with a selective reagent which reacts faster than the conformational changes occur. The use of p mercuribenzoate mercaptide of 2 nitro 5 mercaptobenzoate as an -SH reagent is described to determine K(f) of D glyceralgehyde 3 phosphate dehydrogenase around residue Cys 153, when the hyperreactive side chain Cys 149 was previously carboxymethylated (CM enzyme). The values of K(f) are 0.034 ± 0.005 and 0.014 ± 0.003 in the case of CM apoenzyme and holoemzyme, respectively, in Tris HCl buffer, I = 0.05, pH 7.5, at 5°C. The free enthalpy change (ΔG) accompanying the conformational change which makes Cys 153 accessible to the reagent, is 1.86 ± 0.08 and 2.34 ± 0.11 kcal/mol for CM apoenzyme and holoenzyme, respectively. The enthalpy change (ΔH) is + 3.9 ± 0.7 kcal/mol both in the absence and presence of coenzyme. The entropy change (ΔS) is + 7.6 ± 1.3 and + 6.3 ± 1.1 calXdegree-1Xmol-1 for CM apoenzyme and holoenzyme, respectively. Based on the value of K(f) and the earlier kinetic data for mercaptide formation of Cys 153 one can characterize the structural motility: the rate constants of exposure and masking of residue Cys 153 of the apoenzyme are K(+1) = 2.2 ± 0.2 min-1 and K(-1) = 65 ± 16 min-1 respectively, under the experimental conditions.

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