Open AccessRefolding and Reactivation of Escherichia coli Tryptophan Synthase β 2 Subunit after Inactivation and Dissociation in Guanidine Hydrochloride at Acidic pH
Author(s) -
GROHA Claudia,
BARTHOLMES Peter,
JAENICKE Rainer
Publication year - 1978
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1978.tb12764.x
Subject(s) - guanidine , chemistry , dimer , tryptophan synthase , kinetics , monomer , protein subunit , dissociation (chemistry) , ultracentrifuge , hydrochloride , escherichia coli , enzyme , trimer , reaction rate constant , enzyme kinetics , native state , protein quaternary structure , stereochemistry , crystallography , chromatography , biochemistry , active site , organic chemistry , physics , quantum mechanics , gene , polymer
The isolated β 2 subunit of Escherichia coli tryptopnan synthase can be reversibly deactivated and dissociated in the presence of 4.5 M guanidine hydrochloride at pH 2.3. Using gel chromatography and ultracentrifugation, the denatured state is found to be the homogeneous, inactive monomer ( M r = 44000, s 20,w = 1.7 S). Removal of the denaturant by dilution leads to 90 ± 3% regain of specific activity and complete recovery of the enzymatic, hydrodynamic, and spectral properties characterizing the native, dimeric quanternary structure. The recovery of enzymatic activity obeys first‐order kinetics over a concentration range of 120.0–0.5 μg/ml (3–0.01 μM). However, a full description of the kinetics of reactivation requires the dimerization of the refolding subunits to be included into the reaction scheme. The dimerization has been directly established by time‐dependent measurements of the assembly of the β 2 dimer. Therefore, a sequential uni‐bi‐unimolecular mechanism is proposed, which involves first‐order conformational changes in addition to the second‐order association step. Assuming the monomeric subunits to be inactive, the experimental data can be fitted by one rate constant ( k = 6 ± 1 × 10 −4 s −1 ). This may be ascribed to a slow ‘reshuffling’ process, occurring after the fast association of partially refolded monomers, to form an inactive precursor of the native β 2 dimer.