Irreversible thermal denaturation of uridine phosphorylase from Escherichia coli K-12.

Thermal denaturation of uridine phosphorylase from Escherichia coli K-12 has been studied by differential scanning calorimetry. The excess heat capacity vs. temperature profiles were obtained at temperature scanning rates of 0.25, 0.5, and 1 K/min. These profiles were analysed using three models of irreversible denaturation which are approximations to the whole Lumry-Eyring model, namely, the one-step model of irreversible denaturation, the Lumry-Eyring model with the fast equilibrating first step, and the model involving two consecutive irreversible steps. In terms of statistics the latter model describes the kinetics of thermal denaturation of uridine phosphorylase more satisfactorily than the two other models. The values of energy activation for the first and second steps calculated for the model involving two consecutive irreversible steps are the following: Ea,1 = 609.3 +/- 1.8 kJ/mol and Ea,2 = 446.8 +/- 3.2 kJ/mol.

Denaturation of uridine phosphorylase from Escherichia coli K-12 by guanidine hydrochloride.

Denaturation of uridine phosphorylase from Escherichia coli K-12 by guanidine hydrochloride results in red shift of the maximum in the protein fluorescence spectrum, dissociation of the hexameric enzyme molecule into monomers, and the loss of the enzymatic activity. The initial rate of the enzyme reactivation after the dilution of the enzyme preincubated with guanidine hydrochloride has the second order with respect to protein. It is assumed that the rate of the reactivation process is limited by the reassociation of monomers possessing low enzymatic activity to dimers followed by the rapid step of hexamer formation.

Behavior of uridine phosphorylase from Escherichia coli K-12 in hydrated reversed micelles of surfactant in organic solvent.

The catalytic activity of uridine phosphorylase from Escherichia coli K-12 entrapped in hydrated reversed micelles of aerosol OT (AOT) in octane has been studied as a function of the degree of hydration of micelles. It was shown that the catalytic activity reaches maximum values at ratios [H2O]/[AOT] equal to 8.4, 12.8, 16.1, and 18.6. On the basis of sedimentation data the conclusion has been made that the maximums of the catalytic activity of uridine phosphorylase correspond to monomeric, dimeric, trimeric, and tetrameric forms of the enzyme.

Use of hydrated reversed micelles of surfactant in organic solvent for stabilization of individual oligomeric forms of uridine phosphorylase from Escherichia coli K-12.

The catalytic activity of uridine phosphorylase from Escherichia coli K-12 entrapped in hydrated reversed micelles of aerosol OT (AOT) in octane has been studied as a function of the degree of hydration of the micelles. It was shown that the catalytic activity of uridine phosphorylase reached maximum values at [H2O/[AOT] ratios equal to 8.4, 12.9, 16.1 and 18.6. Based on the sedimentation data the conclusion has been made that the maxima of the catalytic activity correspond to the monomeric, dimeric, trimeric and tetrameric forms of the enzyme. The measurements of the rate of the enzymatic reaction catalyzed by uridine phosphorylase entrapped in hydrated reversed micelles at various concentrations of AOT indicate that the monomeric enzyme form, in contrast to the trimeric and tetrameric forms, exhibits the membranotropic properties.

Denaturation of uridine phosphorylase from Escherichia coli K-12 with guanidine hydrochloride: kinetics of inactivation, dissociation, and reactivation of the enzyme.

Denaturation of uridine phosphorylase from Escherichia coli K-12 by guanidine hydrochloride is accompanied by the displacement of the maximum in the protein fluorescence spectrum (lambda max) from 331 to 348 nm. The half-maximal change in the lambda max position is observed at 1.18 M guanidine hydrochloride. For this concentration of denaturant, the sedimentation pattern consists of two boundaries, one of which corresponds to the motion of the hexameric enzyme form (s20,w = 8.2 S) and other represents a monomer (s20,w = 2.6 S). In the presence of 2 M guanidine hydrochloride the enzyme moves as a monomer. The kinetics of inactivation of uridine phosphorylase by guanidine hydrochloride are complex (minima and maxima are observed on the kinetic curves). The initial rate of the enzyme reactivation after dilution of the enzyme preincubated with guanidine hydrochloride is second order with respect to protein. It is assumed that the rate of the reactivation process is limited by the reassociation of low-activity monomers into dimers followed by a rapid hexamer formation. The second-order rate constant for the reassociation of the enzyme is 3.0.10(4) M-1.sec-1 (50 mM borate buffer, pH 7.7, containing 100 mM inorganic phosphate; 20 degrees C). Thiol groups become accessible to titration by 5,5'-dithiobis-(2-nitrobenzoic acid) after treatment of uridine phosphorylase with guanidine hydrochloride. Uridine and uracil inhibit the unfolding of the protein globule by guanidine hydrochloride.

[Interaction of metal ions with the carboxyl group of Asp-5 in the active site of uridine phosphorylase from Escherichia coli K-12]. / Vzaimodeistvie ionov metallov s karboksil'noi gruppoi Asp-5 v aktivnom tsentre uridinfosforilazy iz Escherichia coli K-12.

The effects of La3+ ions on enzymatic activity and difference absorption spectra of native and fluorescein isothiocyanate (FITC) modified uridine phosphorylase from E. coli K-12 have been studied. Excess La3+, unlike Ag+, only slightly decreases the enzyme activity but provokes similar changes in the absorption spectra of both native and modified proteins. The Kd value for La3+ ions (0.2 mM) coincides with that obtained earlier for Ag+. La3+ ions (0.2 mM) have no effect on the rate of the enzyme inactivation by diethylpyrocarbonate or tetranitromethane but increases the rate of its inactivation by Woodward's reagent K (WRK). Binding of La3+ (Kd = 0.2 mM) markedly decreases the thermal stability of the enzyme which increases with a further rise in the La3+ concentration. The values of Kd (0.2 mM) as well as the difference spectra and specific interactions with WRK indicate that one of the ligands interacting with metal ions is the carboxyl group of the Asp-5 residue. According to X-ray analysis data, this residue is involved in the formation of the active center of uridine phosphorylase.