SEARCH · PubMed Health

Results for “GTP Pyrophosphokinase”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

43 records · Page 3Linked to original sources

PubMed2004

Characterization of the Saccharomyces cerevisiae Fol1 protein: starvation for C1 carrier induces pseudohyphal growth.

Tetrahydrofolate (vitamin B9) and its folate derivatives are essential cofactors in one-carbon (C1) transfer reactions and absolutely required for the synthesis of a variety of different compounds including methionine and purines. Most plants, microbial eukaryotes, and prokaryotes synthesize folate de novo. We have characterized an important enzyme in this pathway, the Saccharomyces cerevisiae FOL1 gene. Expression of the budding yeast gene FOL1 in Escherichia coli identified the folate biosynthetic enzyme activities dihydroneopterin aldolase (DHNA), 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK), and dihydropteroate synthase (DHPS). All three enzyme activities were also detected in wild-type yeast strains, whereas fol1Delta deletion strains only showed background activities, thus demonstrating that Fol1p catalyzes three sequential steps of the tetrahydrofolate biosynthetic pathway and thus is the central enzyme of this pathway, which starting from GTP consists of seven enzymatic reactions in total. Fol1p is exclusively localized to mitochondria as shown by fluorescence microscopy and immune electronmicroscopy. FOL1 is an essential gene and the nongrowth phenotype of the fol1 deletion leads to a recessive auxotrophy for folinic acid (5'-formyltetrahydrofolate). Growth of the fol1Delta deletion strain on folinic acid-supplemented rich media induced a dimorphic switch with haploid invasive and filamentous pseudohyphal growth in the presence of glucose and ammonium, which are known suppressors of filamentous and invasive growth. The invasive growth phenotype induced by the depletion of C1 carrier is dependent on the transcription factor Ste12p and the flocullin/adhesin Flo11p, whereas the filamentation phenotype is independent of Ste12p, Tec1p, Phd1p, and Flo11p, suggesting other signaling pathways as well as other adhesion proteins.

Aldehyde-Lyases↗

PubMed2001

Class II recombinant phosphoribosyl diphosphate synthase from spinach. Phosphate independence and diphosphoryl donor specificity.

A recombinant form of spinach (Spinacia oleracea) phosphoribosyl diphosphate (PRPP) synthase isozyme 3 resembling the presumed mature enzyme has been synthesized in an Escherichia coli strain in which the endogenous PRPP synthase gene was deleted, and has been purified to near homogeneity. Contrary to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows an unusual low specificity toward diphosphoryl donors by accepting dATP, GTP, CTP, and UTP in addition to ATP. The kinetic mechanism of the enzyme is an ordered steady state Bi Bi mechanism with K(ATP) and K(Rib-5-P) values of 170 and 110 micrometer, respectively, and a V(max) value of 13.1 micromol (min x mg of protein)(-1). The enzyme has an absolute requirement for magnesium ions, and maximal activity is obtained at 40 degrees C at pH 7.6.

Catalysis↗

PubMed1977

Effect of lowered intracellular ATP and GTP concentrations on purine ribonucleotide synthesis and interconversion.

The effects of lowered intracellular ATP and GTP concentrations on enzymes of purine ribonucleotide synthesis and intercoversion were studied using intact Ehrlich ascites tumor cells. The apparent rates of phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1) and of inosinate dehydrogenase (EC 1.2.1.14) were increased in cells containing lowered purine nucleotide concentrations, but apparent activities of amidophosphoribosyltransferase (EC 2.4.2.14), the purine phosphoribosyltransferases, and other enzymes of purine ribonucleotide interconversion were not affected.

Adenine Phosphoribosyltransferase↗

PubMed2003

Escherichia coli phnN, encoding ribose 1,5-bisphosphokinase activity (phosphoribosyl diphosphate forming): dual role in phosphonate degradation and NAD biosynthesis pathways.

An enzymatic pathway for synthesis of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene encoding PRPP synthase (B. Hove-Jensen, J. Bacteriol. 178:714-722, 1996). The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase. The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its enzymatic activity has not been determined previously. The reaction sequence is ribose 5-phosphate --> ribose 1-phosphate --> ribose 1,5-bisphosphate --> PRPP. Alternatively, the synthesis of ribose 1-phosphate in the first step, catalyzed by phosphopentomutase, can proceed via phosphorolysis of a nucleoside, as follows: guanosine + P(i) --> guanine + ribose 1-phosphate. The ribose 1,5-bisphosphokinase-catalyzed phosphorylation of ribose 1,5-bisphosphate is a novel reaction and represents the first assignment of a specific chemical reaction to a polypeptide required for cleavage of a carbon-phosphorus (C-P) bond by a C-P lyase. The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end. PhnN was purified almost to homogeneity and characterized. The enzyme accepted ATP but not GTP as a phosphoryl donor, and it used ribose 1,5-bisphosphate but not ribose, ribose 1-phosphate, or ribose 5-phosphate as a phosphoryl acceptor. The identity of the reaction product as PRPP was confirmed by coupling the ribose 1,5-bisphosphokinase activity to the activity of xanthine phosphoribosyltransferase in the presence of xanthine, which resulted in the formation of 5'-XMP, and by cochromatography of the reaction product with authentic PRPP.

Adenosine Triphosphate↗

PubMed1993

Molecular characterization of phosphoribosylpyrophosphate synthetase from Leishmania donovani.

The phosphoribosylpyrophosphate synthetase (PRS) enzyme from parasitic protozoa plays a critical role in the acquisition of exogenous purine bases by providing the phosphoribosylpyrophosphate substrate for phosphoribosylation. To characterize a PRS enzyme from parasitic protozoa, the prs gene was isolated from a genomic library of Leishmania donovani DNA. A 1936-bp SalI fragment was sequenced that encompassed an open reading frame of 1113 nucleotides encoding a polypeptide of 371 amino acids and 40 787 Da. After gap alignment, the leishmanial PRS exhibited 40-42% amino acid identity with a variety of mammalian and prokaryotic PRSs. L. donovani PRS also contained an approx. 20-amino acid stretch that was highly homologous to the phosphoribosylpyrophosphate binding domains of mammalian phosphoribosyltransferase enzymes. Two prs-specific transcripts of 2.6 and 2.1 kb were detected by Northern analysis, and Southern blots of genomic DNA implied that the prs locus was not tandemly repeated in the L. donovani genome. PRS activity was detected in L. donovani extracts, and apparent Km values of approx. 30 microM and approx. 1 mM were calculated for ribose-5-phosphate and ATP, respectively. PRS was sensitive to inhibition by AMP and ADP but refractory to IMP, GMP, GTP, CTP, and UTP. The high apparent Km value of the parasite enzyme for ATP and its insensitivity to inhibition by many nucleotides suggested that kinetic differences between the L. donovani and human PRSs could provide an avenue for rational therapeutic manipulation of parasitic disease. The isolation of the L. donovani prs gene now provides an opportunity to genetically dissect the determinants responsible for the function and regulation of this indispensable enzyme of purine and pyrimidine metabolism in a genus of parasitic protozoa.

Amino Acid Sequence↗

PubMed1993

Fibroblast growth factor-dependent metabolism of hypoxanthine via the salvage pathway for purine synthesis in porcine aortic endothelial cells.

In this study we examined the metabolism of hypoxanthine in fibroblast growth factor (FGF)-stimulated porcine aortic endothelial cells (PAEC). Our previous report indicated that hypoxanthine in fetal bovine serum (FBS) was an essential component for both basal and FGF-dependent growth of PAEC (Hayashi et al., Exp Cell Res 185: 217-228, 1989). Besides hypoxanthine, the addition of various purine bases and purine nucleosides, but not xanthine, xanthosine or any pyrimidine metabolites, restored the limited growth of PAEC cultured in medium containing 10% dialyzed FBS in the presence or absence of FGF. The metabolism of [14C]hypoxanthine was compared in PAEC treated with and without FGF. Treatment of PAEC with FGF for 24 hr enhanced the radioactivity incorporation from [14C]hypoxanthine into both the acid-soluble and -insoluble fractions approximately 2-fold. Upon chromatographic analyses of hypoxanthine metabolites in the acid-soluble nucleotide fraction, it was found that in control PAEC hypoxanthine was largely metabolized to IMP, adenine nucleotides and uric acid, whereas in FGF-treated cells it was converted to ATP, ADP, GTP, xanthine and uric acid. The radioactivity of IMP was lowered in FGF-stimulated cells. The addition of FGF to PAEC increased phosphoribosyl pyrophosphate (PRPP) synthetase activity by approximately 8-fold and the PRPP content by approximately 2-fold, but it did not increase hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity or hypoxanthine transport. On the other hand, methotrexate, an inhibitor of de novo synthesis of purine, did not affect the growth of PAEC. Analyses of the rate of [14C]formate incorporation into total purine compounds showed that PAEC had a low capacity to synthesize purines de novo, which was not stimulated by FGF. These data indicate that FGF stimulates the synthesis of PRPP necessary for the salvage synthesis of purine nucleotides in conjunction with purine bases, e.g. hypoxanthine.

Animals↗

PubMed2001

Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure.

Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts ATP, dATP, GTP, CTP, and UTP as diphosphoryl donors. All of these properties are characteristic for class II PRPP synthases. K(m) values for ATP and ribose 5-phosphate are 77 and 48 microM, respectively. Gel filtration reveals a molecular mass of the native enzyme of approximately 110 kD, which is consistent with a homotrimer. Secondary structure prediction shows that spinach PRPP synthase isozyme 4 has a general folding similar to that of Bacillus subtilis class I PRPP synthase, for which the three-dimensional structure has been solved, as the position and extent of helices and beta-sheets of the two enzymes are essentially conserved. Amino acid sequence comparison reveals that residues of class I PRPP synthases interacting with allosteric inhibitors are not conserved in class II PRPP synthases. Similarly, residues important for oligomerization of the B. subtilis enzyme show little conservation in the spinach enzyme. In contrast, residues of the active site of B. subtilis PRPP synthase show extensive conservation in spinach PRPP synthase isozyme 4.

Amino Acid Sequence↗