Selective modification of putative uridine-binding site of uridine phosphorylase from E. coli with fluorescein 5'-isothiocyanate.

A putative uridine-binding site of uridine phosphorylase (EC 2.4.2.3) from E. coli was modified with fluorescein 5'-isothiocyanate (FITC). Treatment with FITC irreversibly inactivates the enzyme (Ki = 1.0 mM, k2 = 0.15 min-1). Under the conditions of 90% inactivation the incorporation of the reagent reaches about 1 mol per mol of the enzyme subunit. Addition of uridine prevents the enzyme inactivation by FITC. In contrast to this, addition of a second substrate phosphate increases the rate of inactivation by 2.3-fold (k2 = 0.34 min-1), but has no effect on the affinity of the reagent to the enzyme. The modified protein retains the ability to bind phosphate but not uridine. According to differential absorption spectroscopy data, the binding of phosphate to the active site of the enzyme is accompanied by conformational changes which may accelerate the inactivation rate. The data presented suggest that in the UPase FITC occupies the putative uridine-binding site, while the phosphate-binding site still retains the ability to interact with the second substrate.

Isolation, crystallization in the macrogravitation field, preliminary X-ray investigation of uridine phosphorylase from Escherichia coli K-12.

Uridine phosphorylase (UPH) from Escherichia coli K-12 has been purified to near homogeneity from a strain harbouring the udp gene, encoding UPH, on a multicopy plasmid. UPH was purified to electrophoretic homogeneity with the specific activity 230 units/mg with a recovery of 80%, yielding 120 mg of enzyme from 3g cells. Crystals of enzyme suitable for X-ray diffraction analysis were obtained in a preparative ultracentrifuge. The packing of the molecules in the crystals may be described by the space group P2(1)2(1)2(1) with the unit cell constants a = 90.4; b = 128.8; c = 136.8 A. There is one molecule per asymmetric unit, Vm = 2.4. These crystals diffract to at least 2.5-2.7 A resolution. The hexameric structure of UPH was directly demonstrated by electron microscopy study and image processing.

Influence of the rho-15 temperature-sensitive (ts) mutation on the expression of the deo-operon in Escherichia coli.

In the rho-15 temperature-sensitive (ts) mutant deo-operon enzymes show no sensitivity to catabolite repression and are not derepressed under the influence of a constitutive regulatory mutation, cytR. These data suggest that intact Rho-protein along with CRP protein is necessary for a catabolite sensitive deo-operon promoter cytP to work. In addition, there are data suggesting that Rho-factor and CRP-protein interact with each other in regulation of the deo-operon. Thus, in studies of the effect of the rho-15 (ts) and crp mutations, maximum deo-enzyme levels have been found in the double rho-15 (ts) crp mutant, and therefore intact Rho-protein in the crp genome or intact CRP-protein on the rho-15 (ts) background seems to be an obstacle for the deoP promoter in the deo-operon. In rho-15 (ts) a relative increase has been observed in the enzyme activity for a distal purine nucleoside phosphorylase gene with respect to a proximal thymidine phosphorylase gene. However in crp, the rho-15 (ts) mutation has no effect on the polarity gradient, that is on the background of impaired CRP protein Rho-factor does not seem to work as a transcription terminator within the operon.