An Approach for the Determination of Equilibrium Constant of Structural Motility

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 ‐glyceraldehyde‐3‐phosphate dehydrogenase around residue Cys‐153, when the hyper‐reactive 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 holoenzyme, respectively, in Tris‐HCl buffer, I = 0.05, pH 7.5, at 5 ° C. The free enthalpy change (Δ G ) 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 (Δ H ) is + 3.9 ± 0.7 kcal/mol both in the absence and presence of coenzyme. The entropy change (Δ S ) 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 K t 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.