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

Mária Vas, László Boross

Research output: Article

20 Citations (Scopus)

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, Kf, 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 Kf of d‐glyceraldehyde‐3‐phosphate dehydrogenase around residue Cys‐153, when the hyper‐reactive side‐chain Cys‐149 was previously carboxymethylated (CM‐enzyme). The values of Kf are 0.034 ± 0.005 and 0.014 ± 0.003 in the case of CM‐apoenzyme and holoenzyme, 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 cal × degree−1× mol−1 for CM‐apoenzyme and holoenzyme, respectively. Based on the value of Kt and our 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
DOIs
Publication statusPublished - ápr. 1974

ASJC Scopus subject areas

  • Biochemistry

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