Literature summary for 2.4.2.2 extracted from

Liu, Y.K.; Lin, T.H.; Liu, P.F.
ATP alters protein folding and function of Escherichia coli uridine phosphorylase (2017), Arch. Biochem. Biophys., 634, 11-20 .

Activating Compound

Activating Compound	Comment	Organism	Structure
additional information	ATP is not necessary for the enzyme's catalytic function	Escherichia coli

Cloned(Commentary)

Cloned (Comment)	Organism
gene udp, recombinant expression in Escherichia coli strain BL21(DE3)	Escherichia coli

General Stability

General Stability	Organism
Escherichia coli uridine phosphorylase is destabilized in the presence of ATP. ATP alters protein folding and function of the enzyme. ATP specifically accelerates the unfolding rate of uridine phosphorylase with no observable effects on the refolding process, ATP binding mechanism analysis. Purified UPase remains folded following treatment with 0 to about 4 M urea, and unfolding occurred from 4 to 6 M urea in the absence of ATP. Partially unfolded intermediate states of UPase accumulate in the presence of ATP. ATP interacts with a transition state close to the folded state.	Escherichia coli

Organism

Escherichia coli uridine phosphorylase is destabilized in the presence of ATP. ATP alters protein folding and function of the enzyme. ATP specifically accelerates the unfolding rate of uridine phosphorylase with no observable effects on the refolding process, ATP binding mechanism analysis. Purified UPase remains folded following treatment with 0 to about 4 M urea, and unfolding occurred from 4 to 6 M urea in the absence of ATP. Partially unfolded intermediate states of UPase accumulate in the presence of ATP. ATP interacts with a transition state close to the folded state.

Escherichia coli

Inhibitors

Inhibitors	Comment	Organism	Structure
5-benzylacyclouridine	BAU	Escherichia coli

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P0C037	pyrimidine/purine nucleoside phosphorylase	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant enzyme from Escherichia coli strain BL21(DE3) by dialysis, anion exchange chromatography, and gel filtration	Escherichia coli

Renatured (Commentary)

Renatured (Comment)	Organism
recombinant enzyme, unfolding equilibrium of UPase in 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5.0 mM MgCl2, 1.0 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and 10 M urea at pH 8.0, 25°C overnight. Monitoring by pulse proteolysis. Cm values are measured by fitting the band intensities on SDS-PAGE with varying urea concentrations to a simple two-state model. Data are fitted to the two-state model in the absence of ATP and to a three-state model in the presence of ATP. Purified UPase remains folded following treatment with 0 to about 4 M urea, and unfolding occurs from 4 to 6 M urea in the absence of ATP. For refolding experiments, 0.4 mg/ml UPase are initially incubated overnight in 20 mM Tris-HCl buffer, pH 8.0, containing 50 mM NaCl, 5.0 mM MgCl2, 1.0 mM TCEP, and 7.5 M urea to induce the unfolded state. Refolding is then performed by diluting samples five times into refolding buffer to reach the following final conditions: 0.08 mg/ml UPase, 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5.0 MgCl2, 1.0 mM TCEP, and 1.5 M urea with or without 2.0 mM ATP. Partially unfolded intermediate states of UPase accumulate in the presence of ATP	Escherichia coli

Renatured (Comment)

Organism

recombinant enzyme, unfolding equilibrium of UPase in 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5.0 mM MgCl2, 1.0 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and 10 M urea at pH 8.0, 25°C overnight. Monitoring by pulse proteolysis. Cm values are measured by fitting the band intensities on SDS-PAGE with varying urea concentrations to a simple two-state model. Data are fitted to the two-state model in the absence of ATP and to a three-state model in the presence of ATP. Purified UPase remains folded following treatment with 0 to about 4 M urea, and unfolding occurs from 4 to 6 M urea in the absence of ATP. For refolding experiments, 0.4 mg/ml UPase are initially incubated overnight in 20 mM Tris-HCl buffer, pH 8.0, containing 50 mM NaCl, 5.0 mM MgCl2, 1.0 mM TCEP, and 7.5 M urea to induce the unfolded state. Refolding is then performed by diluting samples five times into refolding buffer to reach the following final conditions: 0.08 mg/ml UPase, 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5.0 MgCl2, 1.0 mM TCEP, and 1.5 M urea with or without 2.0 mM ATP. Partially unfolded intermediate states of UPase accumulate in the presence of ATP

Escherichia coli

Subunits

Subunits	Comment	Organism
homohexamer	6 * 27000, the functional unit is a hexamer with alpha/beta-fold configuration	Escherichia coli

Synonyms

Synonyms	Comment	Organism
pyrimidine/purine nucleoside phosphorylase	UniProt	Escherichia coli
udp	-	Escherichia coli
UPase	-	Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Escherichia coli

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.3	-	assay at	Escherichia coli

General Information

General Information	Comment	Organism
malfunction	inhibitors, such as 5-benzylacyclouridine (BAU), block the activity of uridine phosphorylase, inducing an increase in the plasma concentrations of uridine. This phenomenon, designated as uridine rescue, benefits the chemotherapeutic effect of 5-fluorouracil on colon cancer	Escherichia coli
metabolism	uridine phosphorylase is a critical enzyme in pyrimidine salvage metabolism	Escherichia coli
physiological function	the enzyme catalyzes reversible phosphorolysis of uridine to uracil and ribose-1-phosphate, and is responsible for maintenance of the uridine concentration in cells. Uridine phosphorylase is ubiquitously expressed in all living organisms, from bacteria to human, supporting an important role in cell survivability	Escherichia coli