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be reconstituted with high yield after dissociation and deactivation in 2 4 M urea [3] or at alkaline pH (Bartholmes, unpublished results).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
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.