Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PP_i). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP_i product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.     

Structural and Kinetic Studies of the Allosteric Transition in Sulfolobus solfataricus Uracil Phosphoribosyltransferase: Permanent Activation by Engineering of the C-Terminus

Uracil phosphoribosyltransferase catalyzes the conversion of 5-phosphoribosyl-α-1-diphosphate (PRPP) and uracil to uridine monophosphate (UMP) and diphosphate (PP_i). The tetrameric enzyme from Sulfolobus solfataricus has a unique type of allosteric regulation by cytidine triphosphate (CTP) and guanosine triphosphate (GTP). Here we report two structures of the activated state in complex with GTP. One structure (refined at 2.8-Å resolution) contains PRPP in all active sites, while the other structure (refined at 2.9-Å resolution) has PRPP in two sites and the hydrolysis products, ribose-5-phosphate and PP_i, in the other sites. Combined with three existing structures of uracil phosphoribosyltransferase in complex with UMP and the allosteric inhibitor cytidine triphosphate (CTP), these structures provide valuable insight into the mechanism of allosteric transition from inhibited to active enzyme. The regulatory triphosphates bind at a site in the center of the tetramer in a different manner and change the quaternary arrangement. Both effectors contact Pro94 at the beginning of a long β-strand in the dimer interface, which extends into a flexible loop over the active site. In the GTP-bound state, two flexible loop residues, Tyr123 and Lys125, bind the PP_i moiety of PRPP in the neighboring subunit and contribute to catalysis, while in the inhibited state, they contribute to the configuration of the active site for UMP rather than PRPP binding. The C-terminal Gly216 participates in a hydrogen-bond network in the dimer interface that stabilizes the inhibited, but not the activated, state. Tagging the C-terminus with additional amino acids generates an endogenously activated enzyme that binds GTP without effects on activity.     

Regulation of pyrimidine biosynthesis by purine and pyrimidine nucleosides in slices of rat tissue

Evidence of the primary sites for the regulation of de novo pyrimidine biosynthesis by purine and pyrimidine nucleosides has been obtained in tissue slices through measurements of the incorporation of radiolabeled precursors into an intermediate and end product of the pathway. Both purine and pyrimidine nucleosides inhibited the incorporation of [¹⁴C]-NaHCO₃ into orotic acid and uridine nucleotides, and the inhibition was found to be reversible upon transferring the tissue slices to a medium lacking nucleoside. The ammonia-stimulated incorporation of [¹⁴C]NaHCO₃ into orotic acid, which is unique to liver slices, was sensitive to inhibition by pyrimidine nucleosides at physiological levels of ammonia, but this regulatory mechanism was lost at toxic levels of ammonia. Adenosine, but not uridine, was found to have the additional effects of inhibiting the conversion of [¹⁴C]orotic acid to UMP and depleting the tissue slices of PRPP. Since PRPP is required as an activator of the first enzyme of the de novo pathway, CPSase II, and a substrate of the fifth enzyme, OPRTase, these results indicate that adenosine inhibits the incorporation of [¹⁴C]NaHCO₃ into orotic acid and the incorporation of [¹⁴C]orotic acid into UMP by depriving CPSase II and OPRTase, respectively, of PRPP. Uridine or its metabolites, on the other hand, appear to control the de novo biosynthesis of pyrimidines through end product inhibition of an early enzyme, most likely CPSase II. We found no evidence of end product inhibition of the conversion of orotic acid to UMP in tissue slices.     

A General Assay Method for Nucleotide Pyrophosphorylases

A general assay method for nucleotide pyrophosphorylases has been investigated. The principle of the method is based on the measurement of consumption rate of 5-phosphoribosylpyrophosphate (PRPP) during the enzyme reaction. In the method, an enzyme preparation for sample was incubated in a reaction mixture containing a purine or pyrimidine base and PRPP for a certain time, and the amounts of PRPP before and after the reaction were determined. The amount of PRPP was determined by an enzymatic method using orotidine-5′-monophosphate (5′-OMP) pyrophosphorylase and 5′-OMP decarboxylase. Nucleotide pyrophosphorylase activity corresponding to each purine or pyrimidine base was determined from the amount of PRPP consumed per unit time.

The present method is generally applicable for determining activities of any kind of nucleotide pyrophosphorylases, and does not need any tedious separation procedure in all cases. Therefore, comparing with the conventional assay methods for nucleotide pyrophosphorylase activities, this method can be said to be much simpler and reliable. As an application of the present method, activities of several nucleotide pyrophosphorylases in Micrococcus glutamicus have been determined.     

A th-1 Mutant of Arabidopsis thaliana Is Defective for a Thiamin-Phosphate-Synthesizing Enzyme: Thiamin Phosphate Pyrophosphorylase

We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 × 10⁻⁷ molar thiamin were used for the examination of the production of thiamin and thiamin monophosphate (TMP) using 4-methyl-5-hydroxyethylthiazole phosphate and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate as substrates. While the wild-type strain formed both thiamin and TMP, the th-1 mutant did not. When TMP was added to the extracts, the th-1 mutant, as well as wild type, produced thiamin. Accordingly, it was concluded that the th-1 mutant was defective in the activity of TMP pyrophosphorylase. Some of the characteristics of the enzyme from the wild-type plant were examined. The optimum temperature for the reaction is 45°C, and the K_m values for the substrates are 2.7 × 10⁻⁶ molar for 4-methyl-5-hydroxyethylthiazole phosphate and 1.8 × 10⁻⁶ molar for 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate.     

Substrate Inhibition of Uracil Phosphoribosyltransferase by Uracil Can Account for the Uracil Growth Sensitivity of Leishmania donovani Pyrimidine Auxotrophs

The pathogenic protozoan parasite Leishmania donovani is capable of both de novo pyrimidine biosynthesis and salvage of pyrimidines from the host milieu. Genetic analysis has authenticated L. donovani uracil phosphoribosyltransferase (LdUPRT), an enzyme not found in mammalian cells, as the focal enzyme of pyrimidine salvage because all exogenous pyrimidines that can satisfy the requirement of the parasite for pyrimidine nucleotides are funneled to uracil and then phosphoribosylated to UMP in the parasite by LdUPRT. To characterize this unique parasite enzyme, LdUPRT was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Kinetic analysis revealed apparent K_m values of 20 and 99 μm for the natural substrates uracil and phosphoribosylpyrophosphate, respectively, as well as apparent K_m values 6 and 7 μm for the pyrimidine analogs 5-fluorouracil and 4-thiouracil, respectively. Size exclusion chromatography revealed the native LdUPRT to be tetrameric and retained partial structure and activity in high concentrations of urea. L. donovani mutants deficient in de novo pyrimidine biosynthesis, which require functional LdUPRT for growth, are hypersensitive to high concentrations of uracil, 5-fluorouracil, and 4-thiouracil in the growth medium. This hypersensitivity can be explained by the observation that LdUPRT is substrate-inhibited by uracil and 4-thiouracil, but 5-fluorouracil toxicity transpires via an alternative mechanism. This substrate inhibition of LdUPRT provides a protective mechanism for the parasite by facilitating purine and pyrimidine nucleotide pool balance and by sparing phosphoribosylpyrophosphate for consumption by the nutritionally indispensable purine salvage process.     

Regulation and Mechanism of Phosphoribosylpyrophosphate Synthetase: Repression by End Products

Phosphoribosylpyrophosphate (PRPP) synthetase participates in the biosynthesis in bacteria of purine nucleotides, pyrimidine nucleotides, tryptophan, and histidine. The regulation of the synthesis of PRPP synthetase in Salmonella typhimurium was studied. Addition of end products to the growth medium, singly or in combination, resulted in small decreases in the specific activity of PRPP synthetase, but levels of the enzyme were never decreased to less than half of those found when the bacteria were grown on minimal medium. Growth of the bacteria on several different carbon sources or starvation for phosphate had little effect on the specific activity of PRPP synthetase. Over-production of histidine in a histidine regulatory mutant, which would be expected to result in a depletion of intracellular PRPP pools, did not alter PRPP synthetase specific activity. PRPP synthetase levels were examined in auxotrophic strains of S. typhimurium that had been starved for the end products of PRPP. In each case derepression of an enzyme in the biosynthetic pathway for the limiting end product was demonstrated. However, only alterations in the levels of pyrimidine bases in the culture medium brought about derepression and repression of PRPP synthetase. Excess pyrimidines do not completely repress the enzyme. Deprivation of exponentially growing cells for pyrimidines by growth of an auxotrophic mutant on media containing orotic acid, which enters the cells slowly, resulted in a 10-fold derepression of PRPP synthetase. Derepression of PRPP synthetase during uracil starvation was prevented by chloramphenicol. The PRPP synthetase activities of extracts from repressed and derepressed cells responded in identical fashion to heat inactivation, cellulose acetate electrophoresis at several pH values, and in kinetic experiments.     

Pyrimidine ribonucleoside catabolic enzyme activities ofPseudomonas pickettii

Pyrimidine ribonucleoside catabolic enzyme activities of the opportunistic pathogenPseudomonas pickettii were examined. Of the pyrimidine and related compounds tested, only dihydrouracil (nitrogen source) and ribose (carbon source) supported growth. Thin-layer chromatographic separation of the uridine and cytidine catabolities produced byP. pickettii extracts indicated that this pseudomonad contained nucleoside hydrolase activity. Its presence was confirmed by enzyme assay. Hydrolase activity was elevated in both glucose- and ribose-grown cells relative to succinate-grown cells. Nucleoside hydrolase activity was depressed when dihydrouracil served as a nitrogen source. Cytosine deaminase activity was present in extracts prepared from succinate-, glucose- or ribose-grown cells when (NH₄)₂SO₄ served as the nitrogen source although cells grown on glucose or ribose exhibited a higher enzyme activity. Cytosine deaminase activity was not detected in extracts prepared from cells grown on dihydrouracil as a nitrogen source. Both dihydropyrimidine dehydrogenase and dihydropyrimidinase activities were measurable inP. pickettii. The dehydrogenase activity was higher with NADH than with NADPH as its nicotinamide cofactor when uracil served as its substrate. Carbon source did not affect dehydrogenase or dihydropyrimidinase activity greatly but both activities were diminished in cells grown on the nitrogen source dihydrouracil.     

Characterisation of a partially purified uracil phosphoribosyltransferase from the opportunistic pathogenCandida albicans

This paper describes for the first time the partial purification and properties of uracil phosphoribosyltransferase (UPRTase) from the yeastCandida albicans. UPRTase was purified 38 fold by acid precipitation, DEAE-Sephacel chromatography and ultrafiltration. Further purification of UPRTase was unsuccessful due to the labile nature of the enzyme and the failure in obtaining satisfactory stabilizing conditions. SDS-PAGE suggested that the enzyme exists as a dimer of two dissimilar subunits with molecular masses of 47 and 38 kDa. The pH optimum for phosphoribosylation was about 7.5 and the optimal Mg⁺⁺ concentration was 2 mM. The kinetics of the enzymes for its substrates, uracil and 5-phosphoribosyl-1-pyrophosphate (PRPP) were determined by measuring initial enzyme velocities over a wide range of concentrations of either substrate at different fixed concentrations of the second substrate. Graphic analysis of the data by Hanes-Woolf plots indicated that the reaction is indistinguishable from a double displacement reaction. Ping pong mechanism has been previously reported for other phosphoribosyltransferases. The enzyme has a low affinity for its substrates (K _m=70.5 and 186 µM for uracil and PRPP, respectively) as compared with those ofE. coli and baker's yeast. Inhibition studies indicate that 5-fluorouracil acts as an alternative substrate for UPRTase with 1.6 times higher specific activity.Abbreviations UPRTase Uracil phosphoribosyltranferase - PRTases phosphoribosyltransferases - PRPP 5-phosphoribosyl-1-pyrophosphate - 5-FC 5-fluorocytosine - 5-FU 5-fluorouracil - PEI polyethyleneimine - DTT dithiothreitol - DMSO dimethyl sulphoxide - PMSF phenylmethylsulphonyl fluoride - UMP uridine mono-phosphate     

The structures of Thermoplasma volcanium phosphoribosyl pyrophosphate synthetase bound to ribose-5-phosphate and ATP analogs

Phosphoribosyl pyrophosphate (PRPP) synthetase catalyzes the transfer of the pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium (Tv) was expressed in Escherichia coli, crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs β,γ-methylene ATP and ADP in two monoclinic crystal forms, P2₁. The β,γ-methylene ATP- and the ADP-bound binary structures were determined from crystals grown from ammonium sulfate solutions; these crystals diffracted to 1.8 Å and 1.5 Å resolutions, respectively. Crystals of the ternary complex with ADP-Mg²⁺ and R5P were grown from a polyethylene glycol solution in the absence of sulfate ions, and they diffracted to 1.8 Å resolution; the unit cell is approximately double the size of the unit cell of the crystals grown in the presence of sulfate. The Tv PRPP synthetase adopts two conformations, open and closed, at different stages in the catalytic cycle. The binding of substrates, R5P and ATP, occurs with PRPP synthetase in the open conformation, whereas catalysis presumably takes place with PRPP synthetase in the closed conformation. The Tv PRPP synthetase forms a biological dimer in contrast to the tetrameric or hexameric quaternary structures of the Methanocaldococcus jannaschii and Bacillus subtilis PRPP synthetases, respectively.     

Allosteric properties of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermoacidophilic archaeon Sulfolobus solfataricus

The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when assayed at 60 degrees C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. GTP caused an approximately 20-fold increase in the turnover number kcat and raised the Km values for 5-phosphoribosyl-1-diphosphate (PRPP) and uracil by two- and >10-fold, respectively. The inhibition by CTP was complex as it depended on the presence of the reaction product UMP. Neither CTP nor UMP were strong inhibitors of the enzyme, but when present in combination their inhibition was extremely powerful. Ligand binding analyses showed that GTP and PRPP bind cooperatively to the enzyme and that the inhibitors CTP and UMP can be bound simultaneously (KD equal to 2 and 0.5 microm, respectively). The binding of each of the inhibitors was incompatible with binding of PRPP or GTP. The data indicate that UPRTase undergoes a transition from a weakly active or inactive T-state, favored by binding of UMP and CTP, to an active R-state, favored by binding of GTP and PRPP.     

Regulation of the Pyrimidine Biosynthetic Pathway in <Emphasis Type="Italic">Pseudomonas mucidolens</Emphasis>

Control of pyrimidine biosynthesis was examined in Pseudomonas mucidolens ATCC 4685 and the five de novo pyrimidine biosynthetic enzyme activities unique to this pathway were influenced by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. When uracil was supplemented to glucose-grown ATCC 4685 cells, activities of four de novo enzymes were depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the glucose-grown mutant strain cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase activities to increase by more than 3-fold while OMP decarboxylase activity increased by 2.7-fold. The syntheses of the de novo enzymes appeared to be regulated by pyrimidines. At the level of enzyme activity, aspartate transcarbamoylase activity in P. mucidolens ATCC 4685 was subject to inhibition at saturating substrate concentrations. Transcarbamoylase activity was strongly inhibited by UTP, ADP, ATP, GTP and pyrophosphate.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : The Purification and Properties of Farnesyl Transferase from Elicited Seedlings

Farnesyl transferase (farnesyl pyrophosphate: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400-fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 ± 3,000 from its behavior on a calibrated G-100 Sephadex molecular sieving column. Mg²⁺ ion at 4 millimolar gave the greatest stimulation of activity; Mn²⁺ ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (K_m = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K_m = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K_m = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes—a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates.     

Biosynthesis of monoterpenes: partial purification and characterization of 1,8-cineole synthetase from Salvia officinalis.

The heterocyclic monoterpene 1,8-cineole is one of the major components of the volatile oil produced by sage (Salvia officinalis), and soluble enzyme extracts prepared from young sage leaves catalyzed the anaerobic conversion of the acyclic precursor neryl pyrophosphate to 1,8-cineole. This enzymatic activity was partially purified by a combination of ammonium sulfate precipitation and chromatography on hydroxylapatite, and the bulk of the competing activities, including phosphatases, were removed from the preparation. Cineole synthetase activity had a pH optimum at 6.1. The rate of 1,8-cineole formation was linear up to 1 h, and up to a protein concentration of 450 μg/ml. A divalent cation was required for catalysis, and maximum activity was obtained with MnCl₂ (1 mm). ZnCl₂ was nearly as effective as MnCl₂, and MgCl₂ could substitute for MnCl₂ only at tenfold higher concentrations. The apparent K_m and V of the enzyme were 10^?5m and 5.6 nmol/h-mg-ml, respectively. Inhibition of activity was observed at neryl pyrophosphate concentrations above 2 × 10^?4m. Nerol, neryl phosphate, 6,7-dihydroneryl pyrophosphate, citronellyl pyrophosphate, and 3,7-dimethyloctyl pyrophosphate were inactive as substrates for 1,8-cineole biosynthesis, indicating that the pyrophosphate and both double bonds of neryl pyrophosphate were required for catalysis. Geranyl pyrophosphate and linaloyl pyrophosphate were converted to 1,8-cineole at only 9 and 15%, respectively, of the rate of neryl pyrophosphate. Thus, the enzyme was highly specific for neryl pyrophosphate. α-Terpineol and its phosphorylated derivatives were not converted to 1,8-cineole, and this observation, coupled with the resolution of cineole synthetase activity from α-terpineol synthetase activity, proved conclusively that α-terpineol was not an intermediate in 1,8-cineole biosynthesis. p-Hydroxymercuribenzoate strongly inhibited the conversion of neryl pyrophosphate to 1,8-cineole (90% inhibition at 4 × 10^?5m); however, other thiol-directed reagents such as N-ethylmaleimide were much less effective. The enzyme was insensitive to NaF and to several other metabolic inhibitors. This is the first report on the properties of cineole synthetase, a novel enzyme which catalyzes both a carbocyclization and a heterocyclization.     

Partial Purification and Characterization of UDP-Glucose Pyrophosphorylase from Immature Grains of Wheat (Triticum aestivum L.)

Uridine diphosphate glucose pyrophosphorylase (UDP-Glc PPase, EC2.7.7.9) was purified 65 fold from immature grains of wheat (Triticum aestivum L. cv, WH-147) by ammonium sulphate fractionation, DEAE-cellulose anion exchange chromatography and Sephadex G-100 permeation chromatography. The partially purified enzyme, having molecular weight of 72 kD, exhibited broad pH optimum between 8 and 9 and was stable at 4°C for 15 days. At pH 8.5, the enzyme followed typical hyperbolic kinetics with respect to UDP-glucose and inorganic pyrophosphate (Km 0.22 mM and 0.66 mM respectively). The enzyme showed absolute requirement for Mg²⁺ and did not appear to require sulfhydryl groups for its activity. Initial velocity and product inhibition studies indicated sequential addition of substrates and sequential release of products.     

Production of uridine 5′-monophosphate by Corynebacterium ammoniagenes ATCC 6872 using a statistically improved biocatalytic process

Attempts were made with success to develop a two-step biocatalytic process for uridine 5′-monophosphate (UMP) production from orotic acid by Corynebacterium ammoniagenes ATCC 6872: the strain was first cultivated in a high salt mineral medium, and then cells were harvested and used as the catalyst in the UMP production reaction. Effects of cultivation and reaction conditions on UMP production were investigated. The cells exhibited the highest biocatalytic ability when cultivated in a medium containing corn steep liquor at pH 7.0 for 15 h in the exponential phase of growth. To optimize the reaction, both “one-factor-at-a-time” method and statistical method were performed. By “one-factor-at-a-time” optimization, orotic acid, glucose, phosphate ion (equimolar KH₂PO₄ and K₂HPO₄), MgCl₂, Triton X-100 were shown to be the optimum components for the biocatalytic reaction. Phosphate ion and C. ammoniagenes cell were furthermore demonstrated as the most important main effects on UMP production by Plackett–Burman design, indicating that 5-phosphoribosyl-1-pyrophosphate (PRPP) synthesis was the rate-limiting step for pyrimidine nucleotides production. Optimization by a central composition design (CCD) was then performed, and up to 32 mM (10.4 g l⁻¹) UMP was accumulated in 24 h from 38.5 mM (6 g l⁻¹) orotic acid. The yield was threefold higher than the original UMP yield before optimization.     

Pyrophosphate-dependent 6-phosphofructokinase, a new glycolytic enzyme in pineapple leaves.

Pineapple leaves contain a pyrophosphate-dependent 6-phosphofructokinase which has been partially purified and characterized. In crude extracts the pyrophosphate-dependent activity is 10 to 20-fold higher than the ATP-dependent activity. The partially purified activity is near 2.5 μmol Fru-1,6-P₂ formed/min/mg protein. In the reaction 1 Fru-1,6-P₂ is formed per 1 pyrophosphate consumed. The enzyme exhibits a pH optimum of 8.0 and the activity is stimulated by Mg⁺⁺. The discovery of a pyrophosphate-dependent 6-phosphofructokinase in pineapple leaves indicates pyrophosphate can serve as an energy source for synthetic reactions in pineapple and perhaps in other plants as well.     

Pool and synthesis of phosphoribosylpyrophosphate in rat embryo cells infected with X14 or H-1 parvovirus

In rat embryo cell cultures infected with X14 or H-1 parvovirus the PRPP pool and the PRPP synthetase activity have been assayed. A radiometric method, prepared by Authors, based on the conversion of [6-14C) orotate to [6-14C) UMP by the mixed enzyme orotate phosphoribosyltransferase and orotidylate decarboxylase and on the separation of UMP by ascending chromatography, has been utilized. The PRPP pool and te PRPP synthetase activity appeared nearly unmodified in the cells infected with X14 or H-1 parvovirus compared to the mock-infected cells. Therefore, the lowered pyrimidine nucleotide synthesis in infected cells, shown in previous studies, may depend, rather than on the diminished PRPP pool, on the lower PRPP utilization; in fact, some inhibition by metabolites, that may be removed by added PRPP, might occur in the infected cells.     

6-azauracil-resistant variants of cultured plant cells lack uracil phosphoribosyltransferase activity

6-Azauracil-resistant variants of Haplopappus gracilis (Nutt.) Gray and Datura innoxia Mill. lack activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme that catalyzes the conversion of uracil and 6-azauracil to uridine-5′-monophosphate and 6-azauridine-5′-monophosphate, respectively. Resistant cells are competent to take up uracil from their growth medium but do not convert it into a form that can be used for macromolecular synthesis. In extracts from resistant cells, orotate monophosphate decarboxylase, a target enzyme of 6-azauridine monophosphate, is fully sensitive to the phosphorylated analog. These results strongly suggest that uracil phosphoribosyltransferase is the major pathway of pyrimidine salvage in cells of these species and that loss of this enzyme activity confers on the variants resistance to 6-azauracil.