Phosphorolysis Of Uridine Research Articles

Anion exchange resins in phosphate form as versatile carriers for the reactions catalyzed by nucleoside phosphorylases.

In the present work, we suggested anion exchange resins in the phosphate form as a source of phosphate, one of the substrates of the phosphorolysis of uridine, thymidine, and 1-(β-ᴅ-arabinofuranosyl)uracil (Ara-U) catalyzed by recombinant E. coli uridine (UP) and thymidine (TP) phosphorylases. α-ᴅ-Pentofuranose-1-phosphates (PF-1Pis) obtained by phosphorolysis were used in the enzymatic synthesis of nucleosides. It was found that phosphorolysis of uridine, thymidine, and Ara-U in the presence of Dowex® 1X8 (phosphate; Dowex-nPi) proceeded smoothly in the presence of magnesium cations in water at 20–50 °C for 54–96 h giving rise to quantitative formation of the corresponding pyrimidine bases and PF-1Pis. The resulting PF-1Pis can be used in three routes: (1) preparation of barium salts of PF-1Pis, (2) synthesis of nucleosides by reacting the crude PF-1Pi with an heterocyclic base, and (3) synthesis of nucleosides by reacting the ionically bound PF-1Pi to the resin with an heterocyclic base. These three approaches were tested in the synthesis of nelarabine, kinetin riboside, and cladribine with good to excellent yields (52–93%).

Substrate specificity of E. coli uridine phosphorylase. Further evidences of high-syn conformation of the substrate in uridine phosphorolysis

ABSTRACTTwenty five uridine analogues have been tested and compared with uridine with respect to their potency to bind to E. coli uridine phosphorylase. The kinetic constants of the phosphorolysis reaction of uridine derivatives modified at 2′-, 3′- and 5′-positions of the sugar moiety and 2-, 4-, 5- and 6-positions of the heterocyclic base were determined. The absence of the 2′- or 5′-hydroxyl group is not crucial for the successful binding and phosphorolysis. On the other hand, the absence of both the 2′- and 5′-hydroxyl groups leads to the loss of substrate binding to the enzyme. The same effect was observed when the 3′-hydroxyl group is absent, thus underlining the key role of this group. Our data shed some light on the mechanism of ribo- and 2′-deoxyribonucleoside discrimination by E. coli uridine phosphorylase and E. coli thymidine phosphorylase. A comparison of the kinetic results obtained in the present study with the available X-ray structures and analysis of hydrogen bonding in the enzyme-substrate complex demonstrates that uridine adopts an unusual high-syn conformation in the active site of uridine phosphorylase.

Structural investigation of the thymidine phosphorylase from Salmonella typhimurium in the unliganded state and its complexes with thymidine and uridine.

Highly specific thymidine phosphorylases catalyze the phosphorolytic cleavage of thymidine, with the help of a phosphate ion, resulting in thymine and 2-deoxy-α-D-ribose 1-phosphate. Thymidine phosphorylases do not catalyze the phosphorolysis of uridine, in contrast to nonspecific pyrimidine nucleoside phosphorylases and uridine phosphorylases. Understanding the mechanism of substrate specificity on the basis of the nucleoside is essential to support rational drug-discovery investigations of new antitumour and anti-infective drugs which are metabolized by thymidine phosphorylases. For this reason, X-ray structures of the thymidine phosphorylase from Salmonella typhimurium were solved and refined: the unliganded structure at 2.05 Å resolution (PDB entry 4xr5), the structure of the complex with thymidine at 2.55 Å resolution (PDB entry 4yek) and that of the complex with uridine at 2.43 Å resolution (PDB entry 4yyy). The various structural features of the enzyme which might be responsible for the specificity for thymidine and not for uridine were identified. The presence of the 2'-hydroxyl group in uridine results in a different position of the uridine furanose moiety compared with that of thymidine. This feature may be the key element of the substrate specificity. The specificity might also be associated with the opening/closure mechanism of the two-domain subunit structure of the enzyme.

Concerted action of two subunits of the functional dimer of Shewanella oneidensis MR-1 uridine phosphorylase derived from a comparison of the C212S mutant and the wild-type enzyme.

Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The structure of the C212S mutant of uridine phosphorylase from the facultatively aerobic Gram-negative γ-proteobacterium Shewanella oneidensis MR-1 (SoUP) was determined at 1.68 Å resolution. A comparison of the structures of the mutant and the wild-type enzyme showed that one dimer in the mutant hexamer differs from all other dimers in the mutant and wild-type SoUP (both in the free form and in complex with uridine). The key difference is the `maximum open' state of one of the subunits comprising this dimer, which has not been observed previously for uridine phosphorylases. Some conformational features of the SoUP dimer that provide access of the substrate into the active site are revealed. The binding of the substrate was shown to require the concerted action of two subunits of the dimer. The changes in the three-dimensional structure induced by the C212S mutation account for the lower affinity of the mutant for inorganic phosphate, while the affinity for uridine remains unchanged.

High-syn conformation of uridine and asymmetry of the hexameric molecule revealed in the high-resolution structures of Shewanella oneidensis MR-1 uridine phosphorylase in the free form and in complex with uridine.

Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Expression of UP from Shewanella oneidensis MR-1 (SoUP) was performed in Escherichia coli. The high-resolution X-ray structure of SoUP was solved in the free form and in complex with uridine. A crystal of SoUP in the free form was grown under microgravity and diffracted to ultrahigh resolution. Both forms of SoUP contained sulfate instead of phosphate in the active site owing to the presence of ammonium sulfate in the crystallization solution. The latter can be considered as a good mimic of phosphate. In the complex, uridine adopts a high-syn conformation with a nearly planar ribose ring and is present only in one subunit of the hexamer. A comparison of the structures of SoUP in the free form and in complex with the natural substrate uridine showed that the subunits of the hexamer are not identical, with the active sites having either an open or a closed conformation. In the monomers with the closed conformation, the active sites in which uridine is absent contain a glycerol molecule mimicking the ribose moiety of uridine.

The Pyrimidine Nucleoside Phosphorylase of Mycoplasma hyorhinis and How It May Affect Nucleoside-Based Therapy

Mycoplasmas are opportunistic parasites and some species are suggested to preferentially colonize tumor tissue in cancer patients. We could demonstrate that the annotated thymidine phosphorylase (TP) gene in the genome of Mycoplasma hyorhinis encodes a pyrimidine nucleoside phosphorylase (PyNPHyor) that not only efficiently catalyzes thymidine but also uridine phosphorolysis. The kinetic characteristics of PyNPHyor-catalyzed nucleoside and nucleoside analogue (NA) phosphorolysis were determined. We demonstrated that the expression of such an enzyme in mycoplasma-infected cell cultures dramatically alters the activity of various anticancer/antiviral NAs such as 5-halogenated pyrimidine nucleosides, including 5-trifluorothymidine (TFT). Due to their close association with human cancers, the presence of mycoplasmas may markedly influence the therapeutic efficiency of nucleoside-based drugs.

Crystallization of uridine phosphorylase from Shewanella oneidensis MR-1 in the laboratory and under microgravity and preliminary X-ray diffraction analysis.

Uridine phosphorylase (UDP, EC 2.4.2.3), a key enzyme in the pyrimidine salvage pathway, catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The gene expression of UDP from Shewanella oneidensis MR-1 was performed in the recipient strain Escherichia coli. The UDP protein was crystallized on earth (in the free form and in complex with uridine as the substrate) by the hanging-drop vapour-diffusion method at 296 K and under microgravity conditions (in the free form) aboard the Russian Segment of the International Space Station by the capillary counter-diffusion method. The data sets were collected to a resolution of 1.9 Å from crystals of the free form grown on earth, 1.6 Å from crystals of the complex with uridine and 0.95 Å from crystals of the free form grown under microgravity. All crystals belong to the space group P2(1) and have similar unit-cell parameters. The crystal of uridine phosphorylase grown under microgravity diffracted to ultra-high resolution and gave high-quality X-ray diffraction data.

Uridine Phosphorylase from Trypanosoma cruzi: Kinetic and Chemical Mechanisms

The reversible phosphorolysis of uridine to generate uracil and ribose 1-phosphate is catalyzed by uridine phosphorylase and is involved in the pyrimidine salvage pathway. We define the reaction mechanism of uridine phosphorylase from Trypanosoma cruzi by steady-state and pre-steady-state kinetics, pH-rate profiles, kinetic isotope effects from uridine, and solvent deuterium isotope effects. Initial rate and product inhibition patterns suggest a steady-state random kinetic mechanism. Pre-steady-state kinetics indicated no rate-limiting step after formation of the enzyme-products ternary complex, as no burst in product formation is observed. The limiting single-turnover rate constant equals the steady-state turnover number; thus, chemistry is partially or fully rate limiting. Kinetic isotope effects with [1'-(3)H]-, [1'-(14)C]-, and [5'-(14)C,1,3-(15)N(2)]uridine gave experimental values of (α-T)(V/K)(uridine) = 1.063, (14)(V/K)(uridine) = 1.069, and (15,β-15)(V/K)(uridine) = 1.018, in agreement with an A(N)D(N) (S(N)2) mechanism where chemistry contributes significantly to the overall rate-limiting step of the reaction. Density functional theory modeling of the reaction in gas phase supports an A(N)D(N) mechanism. Solvent deuterium kinetic isotope effects were unity, indicating that no kinetically significant proton transfer step is involved at the transition state. In this N-ribosyl transferase, proton transfer to neutralize the leaving group is not part of transition state formation, consistent with an enzyme-stabilized anionic uracil as the leaving group. Kinetic analysis as a function of pH indicates one protonated group essential for catalysis and for substrate binding.

The Crystal Structure of Streptococcus pyogenes Uridine Phosphorylase Reveals a Distinct Subfamily of Nucleoside Phosphorylases

Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 Å resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an α/β monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is ∼7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.

Transition-State Analysis of Trypanosoma cruzi Uridine Phosphorylase-Catalyzed Arsenolysis of Uridine

Uridine phosphorylase catalyzes the reversible phosphorolysis of uridine and 2'-deoxyuridine to generate uracil and (2-deoxy)ribose 1-phosphate, an important step in the pyrimidine salvage pathway. The coding sequence annotated as a putative nucleoside phosphorylase in the Trypanosoma cruzi genome was overexpressed in Escherichia coli , purified to homogeneity, and shown to be a homodimeric uridine phosphorylase, with similar specificity for uridine and 2'-deoxyuridine and undetectable activity toward thymidine and purine nucleosides. Competitive kinetic isotope effects (KIEs) were measured and corrected for a forward commitment factor using arsenate as the nucleophile. The intrinsic KIEs are: 1'-(14)C = 1.103, 1,3-(15)N(2) = 1.034, 3-(15)N = 1.004, 1-(15)N = 1.030, 1'-(3)H = 1.132, 2'-(2)H = 1.086, and 5'-(3)H(2) = 1.041 for this reaction. Density functional theory was employed to quantitatively interpret the KIEs in terms of transition-state structure and geometry. Matching of experimental KIEs to proposed transition-state structures suggests an almost synchronous, S(N)2-like transition-state model, in which the ribosyl moiety possesses significant bond order to both nucleophile and leaving groups. Natural bond orbital analysis allowed a comparison of the charge distribution pattern between the ground-state and the transition-state models.

Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications.

Glycal Formation in Crystals of Uridine Phosphorylase,

Uridine phosphorylase is a key enzyme in the pyrimidine salvage pathway. This enzyme catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate (or 2'-deoxyuridine to 2'-deoxyribose 1-phosphate). Here we report the structure of hexameric Escherichia coli uridine phosphorylase treated with 5-fluorouridine and sulfate and dimeric bovine uridine phosphorylase treated with 5-fluoro-2'-deoxyuridine or uridine, plus sulfate. In each case the electron density shows three separate species corresponding to the pyrimidine base, sulfate, and a ribosyl species, which can be modeled as a glycal. In the structures of the glycal complexes, the fluorouracil O2 atom is appropriately positioned to act as the base required for glycal formation via deprotonation at C2'. Crystals of bovine uridine phosphorylase treated with 2'-deoxyuridine and sulfate show intact nucleoside. NMR time course studies demonstrate that uridine phosphorylase can catalyze the hydrolysis of the fluorinated nucleosides in the absence of phosphate or sulfate, without the release of intermediates or enzyme inactivation. These results add a previously unencountered mechanistic motif to the body of information on glycal formation by enzymes catalyzing the cleavage of glycosyl bonds.

The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy

Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate (R1P). The human UP type 1 (hUP1) is a molecular target for the design of inhibitors intended to boost endogenous uridine levels to rescue normal tissues from the toxicity of fluoropyrimidine nucleoside chemotherapeutic agents, such as capecitabine and 5-fluorouracil. Here, we describe a method to obtain homogeneous recombinant hUP1, and present initial velocity, product inhibition, and equilibrium binding data. These results suggest that hUP1 catalyzes uridine phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate binds first followed by the binding of uridine, and uracil dissociates first, followed by R1P release. Fluorescence titration at equilibrium showed cooperative binding of either P i or R1P binding to hUP1. Amino acid residues involved in either catalysis or substrate binding were proposed based on pH-rate profiles.

Uridine prevents the glucose deprivation-induced death of immunostimulated astrocytes via the action of uridine phosphorylase

We previously reported that in immunostimulated astrocytes, glucose deprivation induced cell death via the loss of ATP, reduced glutathione, and mitochondrial transmembrane potential. The cytotoxicity was due to reactive nitrogen and oxygen species and blocked by adenosine, a purine nucleoside, via the preservation of cellular ATP. Here, we investigated whether uridine, a pyrimidine nucleoside, could prevent the glucose deprivation-induced cytotoxicity in LPS + IFN-γ-treated (immunostimulated) astrocytes. Glucose deprivation induced the death of immunostimulated cells, which was significantly reduced by uridine. Glucose deprivation rapidly decreased cellular ATP levels in immunostimulated astrocytes, which was also reversed by uridine. The inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine attenuated the protective effect of uridine. mRNA and protein expression for uridine phosphorylase, an enzyme catalyzing reversible phosphorolysis of uridine, were observed in rat brain as well as primary astrocytes. 5-(Phenylthio)acyclouridine (PTAU), a specific inhibitor of uridine phosphorylase, inhibited the protective effect of uridine. Additionally, the loss of mitochondrial transmembrane potential and reduced glutathione by glucose deprivation in immunostimulated cells was attenuated by uridine, which was also reversed by PTAU. These results provide the first evidence that uridine protects immunostimulated astrocytes against the glucose deprivation-induced death by preserving intracellular ATP through the action of uridine phosphorylase.

Structural basis for inhibition ofEscherichia coliuridine phosphorylase by 5-substituted acyclouridines

Uridine phosphorylase (UP) catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate and is a key enzyme in the pyrimidine-salvage pathway. Escherichia coli UP is structurally homologous to E. coli purine nucleoside phosphorylase and other members of the type I family of nucleoside phosphorylases. The structures of 5-benzylacyclouridine, 5-phenylthioacyclouridine, 5-phenylselenenylacyclouridine, 5-m-benzyloxybenzyl acyclouridine and 5-m-benzyloxybenzyl barbituric acid acyclonucleoside bound to the active site of E. coli UP have been determined, with resolutions ranging from 1.95 to 2.3 A. For all five complexes the acyclo sugar moiety binds to the active site in a conformation that mimics the ribose ring of the natural substrates. Surprisingly, the terminal hydroxyl group occupies the position of the nonessential 5'-hydroxyl substituent of the substrate rather than the 3'-hydroxyl group, which is normally required for catalytic activity. Until recently, inhibitors of UP were designed with limited structural knowledge of the active-site residues. These structures explain the basis of inhibition for this series of acyclouridine analogs and suggest possible additional avenues for future drug-design efforts. Furthermore, the studies can be extended to design inhibitors of human UP, for which no X-ray structure is available.

Abnormalities in Uridine Homeostatic Regulation and Pyrimidine Nucleotide Metabolism as a Consequence of the Deletion of the Uridine Phosphorylase Gene

We report in the present study the critical role of uridine phosphorylase (UPase) in uridine homeostatic regulation and pyrimidine nucleotide metabolism, employing newly developed UPase-/- mice. Our data demonstrate that the abrogation of UPase activity led to greater than a 6-fold increase in uridine concentrations in plasma, a 5-6-fold increase in lung and gut, and a 2-3-fold increase in liver and kidney, as compared with wild type mice. Urine uridine levels increased 24-fold normal in UPase-/- mice. Uridine half-life and the plasma retention of pharmacological doses of uridine were significantly prolonged. Further, in these UPase-/- mice, abnormal uridine metabolism led to disorders of various nucleotide metabolisms. In the liver, gut, kidney, and lung of UPase-/- mice, total uridine ribonucleotide concentrations increased 2-3 times as compared with control mice. Cytidine ribonucleotides and adenosine and guanosine ribonucleotides also increased, although to a lesser extent, in these organs. Most significant deoxyribonucleotide changes were present in the gut and lung of UPase-/- mice. In these tissues, dTTP concentration increased more than 4-fold normal, and dCTP, dGTP, and dATP concentrations rose 1-2 times normal. In kidney, dTTP concentration increased 2-fold normal, and dCTP and dGTP concentrations rose less than 1-fold normal. In addition, the accumulated uridine in plasma and tissues efficiently reduced 5-fluorouracil host toxicity and altered the anesthetic effect of pentobarbital. These data indicate that UPase is a critical enzyme in the regulation of uridine homeostasis and pyrimidine nucleotide metabolism, and 5-fluorouracil activity.

Mutagenesis of Escherichia coli uridine phosphorylase by random pentapeptide insertions

Escherichia coli uridine phosphorylase (UP) is encoded by the udp gene and catalyzes the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Only few key residues involved in UP catalysis, identified by site-directed mutagenesis and selective chemical modification studies, were reported. A recent paper reporting the crystal structure at 2.0 Å resolution indicated that UP shares a high structural homology with E. coli purine nucleoside phosphorylase. This latter enzyme is better known and a number of residues in its active site have been identified. In this work, we used pentapeptide scanning mutagenesis to cause random insertions of a 5 amino acid cassette in the UP polypeptide chain. Several insertional mutants located in different regions of UP maintained or increased the enzymatic activity and provided new insights into protein structure–function relationships. Moreover, this mutagenesis approach appears to be useful for the rapid preparation of mutants that present altered enzymatic activities.

Characterization of Escherichia coli uridine phosphorylase by single-site mutagenesis.

The Escherichia coli udp gene encodes uridine phosphorylase (UP), which catalyzes the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. The X-ray structure of E. coli UP resolved by two different groups produced conflicting results. In order to cast some light on the E. coli UP catalytic site, we mutagenized several residues in UP and measured by RP-HPLC the phosphorolytic activity of the mutant UP proteins in vitro. Mutations Thr94Ala, Phe162Ala, and Tyr195Gly caused a drastic decrease in UP activity. These three residues were suggested to be involved in the nucleoside binding site. However, surprisingly, Tyr195Ala caused a relative increase in enzymatic activity. Both Met197Ala and Met197Ser conserved low activity, suggesting a minor role for this residue in the UP active site. Glu196Ala completely lost UP activity, whereas the more conservative Glu196Asp mutation was still partially active, confirming the importance of maintaining the correct charge in the surroundings of this position. Glu198 was mutated to either Gly, Asp and Gln. All three substitutions caused complete loss of enzymatic activity suggesting an important role of Glu198 both in ribose binding and in interaction with phosphate ions. Arg30Ala and Arg91Ala eliminated UP activity, whereas Arg30Lys and Arg91Lys presented a very low activity, confirming that these residues might interact with and stabilize the phosphate ions. Ile69Ala did not decrease UP activity, whereas His8Ala lowered the activity to about 20%. Both amino acids were suggested to take part in subunit interactions. Our results confirm the structural similarity between E. coli UP and E. coli purine nucleoside phosphorylase (PNP).

Crystal Structures of Escherichia coli Uridine Phosphorylase in Two Native and Three Complexed Forms Reveal Basis of Substrate Specificity, Induced Conformational Changes and Influence of Potassium

Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway that catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Inhibiting liver UP in humans raises blood uridine levels and produces a protective effect (“uridine rescue”) against the toxicity of the chemotherapeutic agent 5-fluorouracil without reducing its antitumour activity. We have investigated UP–substrate interactions by determining the crystal structures of native Escherichia coli UP (two forms), and complexes with 5-fluorouracil/ribose 1-phosphate, 2-deoxyuridine/phosphate and thymidine/phosphate. These hexameric structures confirm the overall structural similarity of UP to E. coli purine nucleoside phosphorylase (PNP) whereby, in the presence of substrate, each displays a closed conformation resulting from a concerted movement that closes the active site cleft. However, in contrast to PNP where helix segmentation is the major conformational change between the open and closed forms, in UP more extensive changes are observed. In particular a swinging movement of a flap region consisting of residues 224–234 seals the active site. This overall change in conformation results in compression of the active site cleft. Gln166 and Arg168, part of an inserted segment not seen in PNP, are key residues in the uracil binding pocket and together with a tightly bound water molecule are seen to be involved in the substrate specificity of UP. Enzyme activity shows a twofold dependence on potassium ion concentration. The presence of a potassium ion at the monomer/monomer interface induces some local rearrangement, which results in dimer stabilisation. The conservation of key residues and interactions with substrate in the phosphate and ribose binding pockets suggest that ribooxocarbenium ion formation during catalysis of UP may be similar to that proposed for E. coli PNP.

Uridine phosphorylase is a potential prognostic factor in patients with oral squamous cell carcinoma.

Uridine phosphorylase (UPase) catalyzes the reversible phosphorolysis of uridine to uracil. The expression levels and the enzymatic activity of UPase are reported to be higher in human solid tumors than in adjacent normal tissues. However, to the authors' knowledge the clinical significance of UPase expression as determined by immunohistochemical analysis has not been demonstrated in human malignancies. The authors prepared the antibody against UPase, examined the staining of UPase in 72 cases of oral squamous cell carcinoma (SCC) with immunohistochemical analysis using the antibody, and analyzed its relation to clinical and pathologic factors. UPase stained mainly within the invasive edges of tumors and macrophages. UPase-positive staining was observed in 56 of the 72 tumors (77.8%). High staining of UPase (defined as > 50% of cells in a tumor biopsy that are positive for UPase) in primary tumors frequently was associated with the presence of metastasis to lymph nodes (P = 0.007 by the Fischer exact test) and with lower overall survival (P = 0.03 by the log-rank test). The high rate of UPase staining in biopsies from patients with oral SCC may be a prognostic marker for these individuals.