Enzymes Of Purine Metabolism Research Articles

Identification and Characterization of Two Adenosine Phosphorylase Activities in Mycobacterium smegmatis

Purine nucleoside phosphorylase (PNP) is an important enzyme in purine metabolism and cleaves purine nucleosides to their respective bases. Mycobacterial PNP is specific for 6-oxopurines and cannot account for the adenosine (Ado) cleavage activity that has been detected in M. tuberculosis and M. smegmatis cultures. In the current work, two Ado cleavage activities were identified from M. smegmatis cell extracts. The first activity was biochemically determined to be a phosphorylase that could reversibly catalyze adenosine + phosphate ↔ adenine + alpha-D-ribose-1-phosphate. Our purification scheme led to a 30-fold purification of this activity, with the removal of more than 99.9% of total protein. While Ado was the preferred substrate, inosine and guanosine were also cleaved, with 43% and 32% of the Ado activity, respectively. Our data suggest that M. smegmatis expresses two PNPs: a previously described trimeric PNP that can cleave inosine and guanosine only and a second, novel PNP (Ado-PNP) that can cleave Ado, inosine, and guanosine. Ado-PNP had an apparent K(m) (K(m) ( app)) of 98 ± 6 μM (with Ado) and a native molecular mass of 125 ± 7 kDa. The second Ado cleavage activity was identified as 5'-methylthioadenosine phosphorylase (MTAP) based on its biochemical properties and mass spectrometry analysis. Our study marks the first report of the existence of MTAP in any bacterium. Since human cells do not readily convert Ado to Ade, an understanding of the substrate preferences of these enzymes could lead to the identification of Ado analogs that could be selectively activated to toxic products in mycobacteria.

Chemotherapy Effects on the Activities of Some Purine Metabolic Enzymes in Sera of Ovarian Cancer Patients

Adenosine deaminase (ADA; Ec: 3.5.4.4), 5´- Nucleotidase (5´– NT; Ec: 3.1.3.5), and AMP – amino hydrolase (AMP – deaminase AMPDA; Ec: 3.5.4.6) activities were measured in sera of ovarian cancer patients before surgery, and after chemotherapy. The results indicated that ADA specific activity increased significantly (P

Chemotherapy Effects on the Activities of Some Purine Metabolic Enzymes in Sera of Ovarian Cancer Patients

Adenosine deaminase (ADA; Ec: 3.5.4.4), 5´- Nucleotidase (5´– NT; Ec: 3.1.3.5), and AMP – amino hydrolase (AMP – deaminase AMPDA; Ec: 3.5.4.6) activities were measured in sera of ovarian cancer patients before surgery, and after chemotherapy. The results indicated that ADA specific activity increased significantly (P

The role of Zn2+ on the structure and stability of murine adenosine deaminase.

Adenosine deaminase (ADA) is a key enzyme in purine metabolism and crucial for normal immune competence. It is a 40 kDa monomeric TIM-barrel protein containing a tightly bound Zn(2+), which is required for activity. In this study, we have investigated the role of Zn(2+) with respect to ADA structure and stability. After removing Zn(2+), the crystallographic structure of the protein remains highly ordered and similar to that of the holo protein with structural changes limited to regions capping the active site pocket. The stability of the protein, however, is decreased significantly in the absence of Zn(2+). Denaturation with urea shows the midpoint to be about 3.5 M for the apo enzyme, compared with 6.4 M for the holo enzyme. ADA contains four tryptophan residues distant from the Zn(2+) site. (19)F NMR studies in the presence and absence of Zn(2+) were carried out after incorporation of 6-(19)F-tryptophan. Chemical shift differences were observed for three of the four tryptophan residues, suggesting that, in contrast to the X-ray data, Zn(2+)-induced structural changes are propagated throughout the protein. Changes throughout the structure as suggested by the NMR data may explain the lower stability of the Zn(2+)-free protein. Real-time (19)F NMR spectroscopy measuring the loss of Zn(2+) showed that structural changes correlated with the loss of enzymatic activity.

Functional and structural alterations induced by copper in xanthine oxidase

Xanthine oxidase (XO), a key enzyme in purine metabolism, produces reactive oxygen species causing vascular injuries and chronic heart failure. Here, copper's ability to alter XO activity and structure was investigated in vitro after pre-incubation of the enzyme with increasing Cu(2+) concentrations for various periods of time. The enzymatic activity was measured by following XO-catalyzed xanthine oxidation to uric acid under steady-state kinetics conditions. Structural alterations were assessed by electronic absorption, fluorescence, and circular dichroism spectroscopy. Results showed that Cu(2+) either stimulated or inhibited XO activity, depending on metal concentration and pre-incubation length, the latter also determining the inhibition type. Cu(2+)-XO complex formation was characterized by modifications in XO electronic absorption bands, intrinsic fluorescence, and alpha-helical and beta-sheet content. Apparent dissociation constant values implied high- and low-affinity Cu(2+) binding sites in the vicinity of the enzyme's reactive centers. Data indicated that Cu(2+) binding to high-affinity sites caused alterations around XO molybdenum and flavin adenine dinucleotide centers, changes in secondary structure, and moderate activity inhibition; binding to low affinity sites caused alterations around all XO reactive centers including FeS, changes in tertiary structure as reflected by alterations in spectral properties, and drastic activity inhibition. Stimulation was attributed to transient stabilization of XO optimal conformation. Results also emphasized the potential role of copper in the regulation of XO activity stemming from its binding properties.

A 57-gene expression signature in B-cell chronic lymphocytic leukemia

B-CLL is the most frequent type of leukemia in the Western countries. The disease, common among the elderly, follows a variable course in terms of survival time and symptoms. There is evidence that the accumulation of lymphocytes in peripheral blood and bone marrow is due to a cell resistance to apoptosis rather than to highly proliferative cells. Genetic mechanisms that lead to the development and progression of disease are mainly unknown, although a number of prognostically and diagnostically important genetic markers have been identified. The aim of this study is to investigate the gene expression profile, by a specific chip for microarray analysis, in B-CLL lymphocytes with regard to factors involved in apoptosis cascade, signal transduction, purine metabolism enzymes, interleukin expression, enzymes involved in the responses to oxidative stress. We found relevant results in a set of 19 of the 57 genes considered. IMP dehydrogenase, adenine phosphoribosyltransferase, adenylosuccinate lyase, adenylate kinase, ADORA1, G-protein-coupled receptor kinase 6, Bcl-2-like 1 isoform 2, caspase 6, and 8 were found underexpressed; while ADORA3, Gars-Airs-Gart, adenylate kinase 3, adenylate deaminase, NMN adenylyltransferase, CD26, CD38, interleukins 18 and 4 were found overexpressed. The microarray technique is a powerful method for identification of potential important diagnostic and prognostic markers, besides giving prominence to genes candidate for further studies.

Alterations in Adenylate Kinase Activity in Human PBMCs after In Vitro Exposure to Electromagnetic Field: Comparison between Extremely Low Frequency Electromagnetic Field (ELF) and Therapeutic Application of a Musically Modulated Electromagnetic Field (TAMMEF)

This study investigated the effects of electromagnetic fields on enzymes involved in purine metabolism in human peripheral blood mononuclear cells in vitro. Cells were obtained from 20 volunteers. We tested both low-energy, extremely low frequency (ELF; 100-Hz) electromagnetic fields and the Therapeutic Application of Musically Modulated Electromagnetic Fields (TAMMEFs); the latter is characterized by variable frequencies, intensities, and wave shapes. Adenylate kinase activity was increased after ELF field exposure but decreased slightly after TAMMEF exposure. Neither of the two electromagnetic field affected the activities of the purine metabolism enzymes ecto-5′-nucleotidase, adenosine deaminase, and adenosine kinase. We concluded that ELF fields may influence cellular electrical charge stability; stimulation of adenylate kinase activity could restore the cell to a state of equilibrium. In contrast, TAMMEF fields may be useful for maintaining and regulating the cellular electrical charge.

A Phase II Study of the Purine Nucleoside Phosphorylase (PNP) Inhibitor RO5092888 (BCX-4208) in Patients with Moderate to Severe Chronic Plaque Psoriasis: Safety, Tolerability and Lymphocyte Effects

Introduction: PNP is a purine-metabolizing enzyme that catalyzes the phosphorolysis of 2′-deoxyguanosine [dGuo] to guanine and deoxyribose-1-phosphate. In T-cells, PNP inhibition leads to accumulation of deoxyguanosine triphosphate (dGTP), triggering apoptotic cell death. Chronic-plaque type psoriasis (psoriasis) is an autoimmune disorder, in which T-cells contribute, at least in part, to the manifestations and maintenance of the disease. Therefore, targeting T-cells may be a beneficial treatment strategy. Since for chronic inflammatory diseases the safety of potential immunosuppressive drugs is a major consideration, we conducted this study to investigate safety and tolerability of oral RO5092888 (BCX-4208) in patients with moderate to severe chronic plaque psoriasis.Methods: This was a randomized, double-blind, placebo-controlled, dose-ranging study. Sixty-six patients 18 to 70 years old were randomized into one of three groups: placebo, 20mg/d or 120mg/d. The study was conducted from August 2007 to June 2008. Patients received study drug over six weeks and were observed over an additional 4 weeks. Assessments for safety included tracking of adverse events (AEs) including infections; vital signs; ECGs; chemistry panel; LFTs; hematologic parameters including peripheral blood (PB) lymphocyte subsets CD3+, CD4+, CD8+, CD56+, CD20+; and urinalysis.Results: 65 of the 66 enrolled patients were analyzed. One serious AE was observed in the 20mg/d group, a deep vein thrombosis (DVT) in a patient with a history of DVTs, and was considered unrelated to study drug. The percentage of patients experiencing at least one adverse event (AE) of any grade was placebo: 33% (7/21), 20mg: 41% (9/22), and 120mg: 59% (13/22). During treatment, two infections occurred in the placebo group (influenza and sinusitis), one in the 20mg group (nasopharyngitis), and 4 in the 120mg group (2-upper respiratory tract infections, 1 bronchitis, and 1 otitis externa). Reductions in PB lymphocytes and subsets were observed (Table 1). Nine patients showed decreased levels of CD4+ lymphocytes, below 350 cells/μL (20mg/d, 3; 120mg/d, 6).Conclusion: Daily oral administration of 20 mg or 120 mg of RO5092888 for up to 6 weeks demonstrated adequate safety and tolerability. Reductions in PB T cells, T cell subsets and B cells were observed. Further investigation of RO5092888 is warranted in both T-cell and B-cell diseases.Lymphocyte subpopulationMean Nadir (% change from baseline)Placebo (n=21)20 mg (n=22)120 mg (n=22)CD3+24.030.647.5CD4+23.628.944.9CD8+26.131.853.9CD20+37.542.664.2CD56+40.147.772.6Table 1

Molecular Mechanisms of Mitochondrial DNA Depletion Diseases Caused by Deficiencies in Enzymes in Purine and Pyrimidine Metabolism

Mitochondrial DNA depletion syndrome (MDS), a reduction of mitochondrial DNA copy number, often affects muscle or liver. Mutations in enzymes of deoxyribonucleotide metabolism give MDS, for example, the mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) genes. Sixteen TK2 and 22 dGK alterations are known. Their characteristics and symptoms are described. Levels of five key deoxynucleotide metabolizing enzymes in mouse tissues were measured. TK2 and dGK levels in muscles were 5- to 10-fold lower than other nonproliferating tissues and 100-fold lower compared to spleen. Each type of tissue apparently relies on de novo and salvage synthesis of DNA precursors to varying degrees.

Hairy Cell Leukemia: Treatment Successes in the Past 25 Years

Hairy Cell Leukemia: Treatment Successes in the Past 25 Years

Polymorphisms in Folate, Pyrimidine, and Purine Metabolism Are Associated with Efficacy and Toxicity of Methotrexate in Psoriasis

Methotrexate is the gold standard therapy for moderate to severe psoriasis, but there is marked interpersonal variation in its efficacy and toxicity. We hypothesized that in psoriasis patients, specific common polymorphisms in folate, pyrimidine, and purine metabolic enzymes are associated with methotrexate efficacy and/or toxicity. DNA from 203 retrospectively recruited psoriasis patients treated with methotrexate was collected and genotyped by restriction endonuclease digestion or length polymorphism assays. The reduced folate carrier (RFC) 80A allele and the thymidylate synthase (TS) 3'-untranslated region (3'-UTR) 6 bp deletion were associated with methotrexate-induced toxicity (P=0.025 and P=0.025, respectively). RFC 80A and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (ATIC) 347G were associated with methotrexate discontinuation (P=0.048 and P=0.038). The TS 5'-UTR 28 bp 3R polymorphism correlated with poor clinical outcome (P=0.029), however, this was not the case when patients with palmoplantar pustular psoriasis were not included in the analysis. Stronger associations between specific polymorphisms and methotrexate-induced toxicity and discontinuation were found in a subanalysis of patients on methotrexate not receiving folic acid supplementation. We have demonstrated preliminary evidence that specific polymorphisms of enzymes involved in folate, pyrimidine, and purine metabolism could be useful in predicting clinical response to methotrexate in patients with psoriasis.

Substrate analogs induce an intermediate conformational change inToxoplasma gondiiadenosine kinase

Adenosine kinase (AK) is a key enzyme in purine metabolism in the ubiquitous intracellular parasite Toxoplasma gondii and is a potential chemotherapeutic target for the treatment of T. gondii infections. To better understand the structure-activity relationship of 6-substituted purine ribosides, the structures of the T. gondii AK-N6,N6-dimethyladenosine (DMA) complex, the AK-DMA-AMP-PCP complex, the AK-6-methyl mercaptopurine riboside (MMPR) complex and the AK-MMPR-AMP-PCP complex were determined to 1.35, 1.35, 1.75 and 1.75 A resolution, respectively. These structures reveal a conformation intermediate between open and closed, with a small lid-domain rotation of 12 degrees . Residues Gly143-X-X-Gly146 undergo torsional changes upon substrate binding, which together with a Gly68-Gly69 switch induces a hinge bending of the lid domain. The intermediate conformation suggests that ATP binding is independent of adenosine binding. Orienting the gamma-phosphate group of ATP into the optimal catalytic position may be the last step before the onset of chemical catalysis and may require the translocation of Arg136 following the complete closure of the lid domain. 6-Substituted purine-nucleoside analogs are accommodated in a hydrophobic cavity. Modification at the N6 or C6 position of the nucleoside would affect the interactions with the surrounding residues and the binding affinity.

Structure of Zebra fish HIUase: Insights into Evolution of an Enzyme to a Hormone Transporter

During early vertebrate evolution, a duplication event in the gene encoding 5-hydroxyisourate hydrolase (HIUase), a widely distributed enzyme of purine metabolism, gave rise to transthyretin (TTR), a thyroid hormone transporter. We report here on the crystal structure of zebra fish HIUase in two different crystal forms. Despite the phylogenetic distance, this structure compares well with those of newly characterized bacterial HIUases, especially with regard to catalytic regions, which are highly preserved. Comparison with TTR structure reveals a highly conserved scaffold, harbouring distinct functional sites located in the same regions of the two vertebrate proteins. Residues that are differentially conserved in HIUases compared to TTR map in putative catalytic regions occupying significant portions of the two halves of a central channel that transverses the whole TTR protein. The evolution of TTR has been accompanied by remarkable changes of the HIUase active sites that gave rise to a channel open at both ends, thus allowing free access to hormone molecules.

Changes of purine and pyrimidine nucleotide biosynthesis during shoot initiation from epicotyl explants of white spruce (Picea glauca)

Nucleotide metabolism was investigated during white spruce organogenesis by following the metabolic fate of 14C-labeled adenine, adenosine and inosine, as purine precursors, and orotic acid, uridine, and uracil, as pyrimidine intermediates. Key enzymes of purine and pyrimidine metabolism were also assayed during the organogenic process. White spruce epicotyl explants cultured on shoot-forming (SF) medium had a better ability to utilize adenine and adenosine for nucleotide and nucleic acid synthesis, compared to tissue cultured on non-shoot forming (NSF) medium. High levels of salvage products were observed in SF tissue after 10 days in culture, when shoot formation was initiated along the epicotyl axis of the explants. Such a differential utilization of purine precursors was mainly due to the higher specific activity of the two adenine and adenosine salvage enzymes, adenine phosphoribosyltransferase (APRT) and adenosine kinase (AK), measured in SF tissue. Similar catabolism of inosine was observed in both SF and NSF conditions during the 30 days of culture. For pyrimidines, the higher activities of the de novo, salvage, and degradation pathways observed in SF tissue, compared to NSF tissue throughout the course of the experiment, clearly denote a faster turnover of pyrimidine nucleotides in the former. Taken together, these results suggest that a better utilization of purine bases and nucleosides for nucleotide and nucleic acid synthesis, as well as a more rapid turnover of pyrimidine nucleotides, represent a physiological switch, which occurs during the initiation and continuation of the organogenic process in white spruce.

Pentostatin for the Treatment of Indolent Lymphoproliferative Disorders

Purine analogues have been shown to be active in a variety of B- and T-cell malignancies. Among them, pentostatin is also a tight binding inhibitor of adenosine deaminase (ADA), a key enzyme of purine metabolism. ADA is present in all human tissues, with the highest levels in the lymphoid system. Early clinical trials with pentostatin used high doses for acute lymphoblastic leukemias, which were characterized by high levels of ADA. Through the efforts of a few investigators, low-dose regimens that are active and well tolerated for indolent lymphoid malignancies have been developed. Myelosuppressive adverse effects have been shown to be minimal using these schedules. Lymphoplasmacytic lymphoma (LL) is an indolent chronic B-cell lymphoproliferative disorder moderately responsive to alkylating agents. All of the purine analogues have shown activity in LL. However, the advantage of pentostatin over the other agents is the relatively specific toxicity to lymphoid cells and the paucity of myelosuppression as a single agent. No direct comparisons of the agents have been investigated, although pentostatin may be considered to be preferred since it has not been associated with toxicity to myeloid progenitors in colony assays. This is of significance for patients who might benefit from high-dose chemotherapy with autologous stem cell transplantation.

Pyrazolo[4,3-e][1,2,4]triazines: Purine Analogues with Electronic Absorption in the Visible Region

Synthesis of several pryrazolo[4,3-e][1,2,4]-triazines is described. The absorption spectrum of some 5-substituted derivatives was found to extend to the visible region. These compounds were found to inhibit some enzymes of purine metabolism, like xanthine oxidase or bacterial purine-nucleoside phosphorylase with Ki values in the 10-3 – 10-5 M range.

Non-nucleoside adenosine deaminase inhibitors: 2000 – 2004

Adenosine deaminase (ADA) is a crucial enzyme in purine metabolism that irreversibly deaminates adenosine to form inosine. ADA is ubiquitous in human tissues and plays a crucial role in immune system development. ADA inhibitors may be useful for the treatment of ischaemic injury, hypertension, lymphomas and leukaemia. Furthermore, ADA inhibitors have recently also been considered as anti-inflammatory drugs. Most ADA inhibitors to date are purine nucleosides or alkyladenine analogues that have poor pharmacokinetics and/or several toxicities. ADA inhibitors without these problems may improve the treatment of leukaemia and also have potential for use in many other clinical conditions. This hypothesis has generated considerable interest in the development of non-nucleoside ADA inhibitors. Recently, a Fujisawa non-nucleoside ADA inhibitor demonstrated in vivo efficacy in inflammation and lymphoma models after oral administration. This review describes recent advances in the development of non-nucleoside ADA inhibitors, based on the patent literature from January 2000 to December 2004.

Amphioxus allantoicase: Molecular cloning, expression and enzymatic activity

Allantoicase, one of the purine metabolism enzymes, is progressively truncated during the chordate evolution, yet it is unknown when its activity became phylogenetically extinct. In this study, a cDNA encoding allantoicase was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 2441 bp long, and contains an open reading frame encoding a protein of 392 amino acid residues. RT-PCR analysis showed that amphioxus allantoicase was strongly expressed in the hepatic caecum, and weakly expressed in other tissues including hind-gut, gill, muscle, notochord, testis and ovary. The parallel experiment was performed measuring the allantoicase activity in the same tissues revealed that its activity was high in the hepatic caecum, but low or undetectable in other tissues examined. These suggest that allantoicase remains in action in the primitive chordate amphioxus.

Loss of ecto-5′nucleotidase from porcine endothelial cells after exposure to human blood: Implications for xenotransplantation

The endothelial cell surface expression of ecto-5'-nucleotidase (E5'N, CD73) is thought to be essential for the extracellular formation of cytoprotective, anti-thrombotic and immunosuppressive adenosine. Decreased E5'N activity may play a role in xenograft acute vascular rejection, preventing accommodation and tolerance mechanisms. We investigated the extent of changes in E5'N activity and other enzymes of purine metabolism in porcine hearts or endothelial cells when exposed to human blood or plasma and studied the role of humoral immunity in this context. Pig hearts, wild type (WT, n = 6) and transgenic (T, n = 5) for human decay accelerating factor (hDAF), were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were exposed for 3 h to autologous porcine plasma (PP), normal (NHP) or heat inactivated human plasma (HHP), with and without C1-inhibitor. Enzyme activities were measured in heart or endothelial cell homogenates with an HPLC based procedure. The baseline activity of E5'N in WT and T porcine hearts were 6.60 +/- 0.33 nmol/min/mg protein and 8.54 +/- 2.10 nmol/min/mg protein respectively (P < 0.01). Ex vivo perfusion of pig hearts with fresh human blood for 4 h resulted in a decrease in E5'N activity to 4.01 +/- 0.32 and 4.52 +/- 0.52 nmol/min/mg protein (P < 0.001) in WT and T hearts respectively, despite attenuation of hyperacute rejection in transgenic pigs. The initial PAEC activity of E5'N was 9.10 +/- 1.40 nmol/min/mg protein. Activity decreased to 6.76 +/- 0.57 and 4.58 +/- 0.47 nmol/min/mg protein (P < 0.01) after 3 h exposure of HHP and NHP respectively (P < 0.05), whereas it remained unchanged at 9.62 +/- 0.88 nmol/min/mg protein when incubated with PP controls. C1-inhibitor partially preserved E5'N activity, similar to the effect of HHP. Adenosine deaminase, adenosine kinase and AMP deaminase (other enzymes of purine metabolism) showed a downward trend in activity, but none were statistically significant. We demonstrate a specific decrease in E5'N activity in pig hearts following exposure to human blood which impairs adenosine production resulting in a loss of a cytoprotective phenotype, contributing to xenograft rejection. This effect is triggered by human humoral immune responses, and complement contributes but does not fully mediate E5'N depletion.

Selectivity of febuxostat, a novel non-purine inhibitor of xanthine oxidase/xanthine dehydrogenase

The purine analogue, allopurinol, has been in clinical use for more than 30 years as an inhibitor of xanthine oxidase (XO) in the treatment of hyperuricemia and gout. As consequences of structural similarities to purine compounds, however, allopurinol, its major active product, oxypurinol, and their respective metabolites inhibit other enzymes involved in purine and pyrimidine metabolism. Febuxostat (TEI-6720, TMX-67) is a potent, non-purine inhibitor of XO, currently under clinical evaluation for the treatment of hyperuricemia and gout. In this study, we investigated the effects of febuxostat on several enzymes in purine and pyrimidine metabolism and characterized the mechanism of febuxostat inhibition of XO activity. Febuxostat displayed potent mixed-type inhibition of the activity of purified bovine milk XO, with Ki and Ki′ values of 0.6 and 3.1 nM respectively, indicating inhibition of both the oxidized and reduced forms of XO. In contrast, at concentrations up to 100 μM, febuxostat had no significant effects on the activities of the following enzymes of purine and pyrimidine metabolism: guanine deaminase, hypoxanthine-guanine phosphoribosyltransferase, purine nucleoside phosphorylase, orotate phosphoribosyltransferase and orotidine-5′-monophosphate decarboxylase. These results demonstrate that febuxostat is a potent non-purine, selective inhibitor of XO, and could be useful for the treatment of hyperuricemia and gout.