Refolding and Reactivation of Escherichia coli Tryptophan Synthase β₂ Subunit after Inactivation and Dissociation in Guanidine Hydrochloride at Acidic pH

Abstract

The isolated 82 subunit of Eseherichia c d i tryptophan synthase can be reversibly deactivated and dissociated in the presence of 4.5 M guanidine hydrochloride at pH 2.3. Using gel chromatography and ultracentrjfugation, the denatured state is found to be the homogeneous, inactive monomer (Ail, = 44000, . S~O .~ = 1.7 S). Removal of the denaturant by dilution leads to 90 k 3 ?( regain of specific activity and complete recovery of the enzymatic, hydrodynamic, and spectral properties characterizing the native, dimeric quaternary structure.The recovery of enzymatic activity obeys first-order kinetics over a concentration range of 120.0-0.5 pg/ml (3 -0.01 pM). 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 f12 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 k 1 x s-l). 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 p2 dimer.The isolated j32 subunit of the tryptophan synthase M Z [~ multienzyme complex catalyzes the irreversible condensationwhich can be formally considered a partial reaction of the overall schemecatalyzed by the native r& complex. Evidence from recent experiments has proven that the dimer can be reconstituted with high yield after dissociation and deactivation in 2 4 M urea [3] or at alkaline pH (Bartholmes, unpublished results). The aim of the following study is (a) to optimize the reactivation conditions, (b) to characterize the reactivated enzyme and (c) to investigate its niechanism of reconstitution. Experiments refer to the isolated flz dimer in the absence of the corresponding r chains. MATERIALS AND METHODSTryptophan synthase 82 subunit was purified from the A2/F'A2 mutant of Escherichiu coli according to [ l ] and kept frozen at -80 "C in 0.6 M phosphate buffer pH 7.5 + 0.2 mM dithioerythritol + 5 mM EDTA + 0.1 m&l phenylmethylsulfonylfluoride + 0.2 mM pyridoxal 5'-phosphate.Guanidine . HCI was obtained from SchwartzMann (New York), tris(hydroxymethy1)aniinomethane from Roth (Karlsruhe), pyridoxal 5'-phosphate

Purification of Tryptophan Synthase from Saccharomyces cerevisiae and Partial Activity of Its Nicked Subunits

“…Based on earlier reactivation experiments with oligomeric enzymes [34-361 it has been shown recently that E. coli tryptophan synthase f l~ subunit can be reversibly denatured in the presence of 2 4 M urea [37] or 4.5 M guanidine . HCl at acidic pH [38]. Similar observations have been put forward for the corresponding CI subunit [39].…”

Kinetics of appearance of an early immunoreactive species during the refolding of acid-denatured Escherichia coli tryptophan synthase .beta.2 subunit

“…Thus, the experimental value ~2 0 ,~~1 . 5 = 1.9 S found here compares well with the s20,w values reported for P chains in 0.1 M glycine/HCl, pH 2.3, plus 4.5 M GdmHC1 and in 4 M Gdn.HC1, respectively 1.7 S (Groha et al, 1978) and 2 S (C. R. Zetina and M. E. Goldberg, unpublished results),…”

Interactions of non‐detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation

“…The renaturation yield (in%) is shown as a function of the NDSB concentration and was determined as the ratio of the activity of the renatured sample to its theoretical initial activity, obtained from the specific activity of the native enzyme and the total protein concentration (0.5 mg/ml) in the renaturation buffer. previously been observed that Gdn/HCl unfolded β chains can fold efficiently only at low temperatures and low protein concentrations [19]. Unfolded β chains aggregate heavily and renature with extremely low yields (5Ϫ7 %) when folding is achieved at 0.5 mg/ml and 20°C.…”