An improved purification procedure for the beta2 subunit of tryptophan synthase from from Escherichia coli has led to an essentially pure and stable preparation with a specific enzymatic activity that is 30% higher than the previously reported maximum value. Sedimentation analysis shows that the apo-beta2 subunit is monodisperse and dimeric down to a concentration of 0.02 mg of protein/ml. The binding of pyridoxal 5'-phosphate (pyridoxal-P) to the apo-beta2 subunit and to the alpha2-apo-beta2 complex was studied by equilibrium dialysis and spectroscopic titration. Both the beta2 subunit and the alpha2beta2 complex bind 2 mol of pyridoxal-P with no unspecific binding observable at higher concentrations of pyridoxal-P. The binding of pyridoxal-P to the apo-beta2 subunit is cooperative (Hill coefficient nH = 1.7). The data have been fitted to the Adair equation, yielding the apparent microscopic dissociation constants for the complexes with one and two bound ligand molecules. They differ by a factor of 38, suggesting that the apo- and holo-beta2 subunits have distinct conformations. The binding of pyridoxal-P to the alpha2-apo-beta2 complex is noncooperative with a value of the dissociation constant intermediate between the two values of the beta2 subunit. This finding suggests that the alpha subunit may stabilize a third conformational state of the beta2 subunit.
Enolase from Streptococcus mutans has been purified to homogeneity by a three-step procedure. As shown by analytical ultracentrifugation and poly-acrylamide gel electrophoresis under denaturing conditions, the purified enzyme is an octamer with molecular weight Mr = 360 kDa, composed of eight identical subunits with Mr = 45 kDa. The kinetic parameters of S. mutans enolase in the presence of 1 mM Mg2+ are Aspec = 130IU × mg-1 and KM = 0.44 mM as determined by steady-state experiments in the D-glycerate 2-phosphate to enolpyruvate phosphate reaction. Enzymatic activity is inhibited noncompetitively by fluoride in the range between 0 and 10 mM NaF yielding Ki = Ki = 1.39 mM, but inhibition characteristics become competitive when tested above 10 mM. In the presence of only small amounts of phosphate (0.5 mM) the inhibitory effect of fluoride is enhanced dramatically yielding Ki = 0.26 mM. Inhibition by NaPO3F is competitive with Ki = 0.9 mM, indicating that free fluoride ions in combination with phosphate are more effective in inhibiting S. mutans enolase. It can be concluded that inhibition of enolase by fluoride in combination with phosphate can influence glycolysis in S. mutans and so reduce acid production or even growth rate, thereby leading to potential anticariogenic effects.
The mechanism of pryidoxal 5'-phosphate (PLP) binding to both the alpha apo beta 2 complex and the apo beta 2 subunit of tryptophan synthase was investigated by rapid mixing experiments. Absorption and fluorescence changes were used to monitor the binding reaction directly. Reduction with sodium borohydride provided the rate of formation of the internal aldimine with the lysine amino group of the enzyme, and substrate turnover monitored the rate of formation of active enzyme. The alpha 2 apo beta 2 complex binds PLP in a sequence of three steps of decreasing rate: formation of a noncovalent complex, which isomerizes to an enzymically inactive internal aldimine, followed by formation of an active alpha 2 holo beta 2 complex. The two binding sites appear to bind PLP independently. The apo beta 2 subunit binds PLP cooperatively in a sequence of three steps of decreasing rate: formation of a noncovalent complex, which isomerizes to an enzymically inactive internal aldimine, followed by the formation of the enzymically active holo beta 2 subunit. Taken together with kinetic studies of pyridoxine phosphate binding [Tschopp, J., & Kirschner, K. (1980) Biochemistry (second paper of three in this issue)], the rate data of the apo beta 2 subunit are shown to be consistent with the concerted mechanism. The difference between the values of the isomerization rate constants of bound PLP and bound PNP appear to result from the covalent internal aldimine, which is formed with PLP but not with PNP.
Association of the apo-beta 2 and the holo-(beta-PLP)2 subunits of tryptophan synthase from Escherichia coli (L-serine hydro-lyase (adding indole) (EC 4.2.1.20)) with alpha subunits of the same enzyme has been studied by microcalorimetry. The results obtained from thermometric titrations clearly demonstrate that only the native complex alpha2beta 2 is formed, independent of an excess of alpha protein. The reaction of the holo-(beta-PLP)2 with alpha subunits at 25 degrees C is accompanied by a negative enthalpy change, which is almost twice as large as that for complex formation with the apo-beta 2 protein, thus indicating that the interaction enthalpy becomes more favorable in the presence of the coenzyme pyridoxal 5'-phosphate (PLP). Both reaction enthalpies show very large negative temperature coefficients, -3600 +/- 100 cal K-1 (Mol of beta 2)-1 being the value for the formation of the apoenzyme and -2300 +/- 100 cal K-1 (mol of beta 2)-1 pertaining to formation of the holoenzyme. The studies on the association of alpha and beta2 subunits in the two buffers revealed that at 25 degrees C approximately 0.75 proton are absorbed in the presence and absence of the coenzyme, whereas at 35 degrees C one proton is taken up from the solution when PLP is present, but two if the apo-beta 2 complex reacts. These results are a clear indication of energetic linkage between intersubunit interaction, hydrogen ion equilibria, and the binding of the coenzyme.
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
High hydrostatic pressure has been shown to cause reversible dissociation of the isolated apo beta 2 dimer of tryptophan synthase from Escherichia coli into enzymatically inactive monomers [Seifert, T., Bartholmes, P., & Jaenicke, R. (1982) Biophys. Chem. 15, 1-8]. Addition of the coenzyme pyridoxal 5'-phosphate affects the structural stability, as well as the kinetics of dissociation and deactivation. The apo beta 2 dimer is deactivated faster than the holoenzyme by a factor of 10. The midpoints of the corresponding equilibrium transition curves are observed at 690 and 870 bar, respectively. As shown by hybridization of native and chemically modified beta chains, the loss of enzymatic activity is accompanied by subunit dissociation. An additional deactivating effect is produced by the pressure-induced release of the cofactor from the holoenzyme. Renaturation after decompression has been monitored by circular dichroism and intrinsic fluorescence emission. Alterations of the dichroic absorption at 222 nm reflect the recovery of the native secondary structure, while tryptophan fluorescence represents a specific probe for the native tertiary structure in the immediate neighborhood of the active center of the enzyme. By application of both methods to monitor the reconstitution of the apo beta 2 dimer, two first-order processes may be separated along the time scale. The faster phase (k1 = 1.2 X 10(-2) s-1) yields a "structured monomer" with 85% native secondary structure and the tryptophan side chain buried in its native hydrophobic environment. As indicated by sodium borohydride reduction, this intermediate is able to interact with the coenzyme pyridoxal 5'-phosphate in the correct way; however, it does not show enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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