Mechanism of Reactivation and Refolding of Glyceraldehyde‐3‐Phosphate Dehydrogenase from Yeast after Denaturation and Dissociation

Abstract

Glyceraldehyde‐3‐phosphate dehydrogenase from yeast has been reversibly dissociated utilizing various denaturants. Applying comparable experimental conditions, dissociation, denaturation, and deactivation run parallel. Dissociation and reassociation occur at different limiting concentrations of the denaturant causing a range of metastability where irreversible aggregation competes with the process of reconstitution. In order to realize a high yield in kinetic reactivation experiments, solvent conditions far from the tetramer ⇌ monomer transition were chosen. Maximum recovery of enzymatic activity was obtained after denaturation in strong denaturants (citrate pH 2.3 + 6 M guanidine · HCl), and reactivation in the presence of 10 mM NAD++ 0.1–10 mM dithiothreitol at pH 7.6, c < 0.2 μM.Removal of the denaturant by dilution, and separation of high aggregates, e.g. by gelchromatography, lead to 100% regain of specific enzymatic activity and complete recovery of the hydrodynamic and spectral properties characterizing the native tetrameric enzyme.The analysis of the time course of reactivation and renaturation suggests that transconformation of inactive monomers and their subsequent association are responsible for the sigmoidal kinetic traces. A sequential uni‐bimolecular mechanism with first and second order rate constants k1= (11 ± 2) × 10−3 s−1 and k2= 45 ± 10 mM−1· s−1 (15°C) makes it possible to fit the reactivation kinetics in a quantitative manner. Within the limits of error k1 and k2 hold for the reactivation in the absence and in the presence of NAD+. NADH leads to complex kinetic profiles which cannot be fitted by a simple mechanism due to instability of the enzyme in the presence of the reduced coenzyme.