Glycinamide Ribonucleotide Transformylase Research Articles

PURIFICATION OF MAMMALIAN GLYCINAMIDE RIBONUCLEOTIDE (GAR) SYNTHETASE: 80

GAR synthetase, the third enzyme of de novo purine biosynthesis, has been purified to homogeneity from rat, and partially from human and hamster sources. Physiochemical characteristics of the purfied proteins were determined and compared. There is mounting evidence that GAR synthetase is part of a multifunctional protein which includes 5-aminoimidazole ribonucleotide (AIR) synthetase and GAR transformylase activities. In order to answer this question, the activities of these latter two enzymes were monitored during the purification of GAR synthetase. In addition to being part of a multifunctional protein, GAR synthetase is also thought to be a component of a multienzyme complex. Antibodies raised to the purified enzyme were used in conjunction with Western blots to study the molecular weight of GAR synthetase under conditions that would maintain the integrity of any complex present and then under conditions that would lead to the dissociation of the complex into its various components. Finally, antibodies raised to rat liver GAR synthetase were used to select cDNA clones of the gene from a λgtll expression library. These clones will be subsequently used to characterize the structure of the gene encoding GAR synthetase in mammalian cells. This work was supported by grants NIH AG 00029 and NIH HD 13432. This is ERICR contribution #564.

Mammalian glycinamide ribonucleotide transformylase: purification and some properties.

Glycinamide ribonucleotide transformylase, the first of the two formyl group transferases of de novo purine biosynthesis requiring 10-formyltetrahydrofolate, has been purified 1500-fold, nearly to homogeneity, from the murine lymphoma cell line L5178Y. Purification of the enzyme was facilitated by the use of a gelatin protease "affinity" resin. This mammalian enzyme is a monomer of approximate Mr 110 000. The kinetic studies are consistent with a sequential reaction mechanism and yield Michaelis constants of 0.4 mM for the substrate, glycinamide ribonucleotide, and 0.25 microM for the cofactor analogue 10-formyl-5,8-dideazafolate. A minimum Vmax of 2 mumol/(min . mg) was obtained for the purified enzyme, from which a turnover number of 4 s-1 was calculated.

A Drosophila metabolic gene transcript is alternatively processed

We have determined the organization and transcription of a Drosophila DNA segment coding for the purine pathway enzyme, GAR transformylase. Because the transcript encoding this enzyme is very rare, we used a novel method for exon mapping without isolating a cDNA. Our results suggest a long polypeptide having multiple domains, with GAR transformylase at the COOH terminus preceded by an extensive repeat. Part of the same DNA segment specifies a shorter transcript, which consists of the same 5′ exons but lacks the last three 3′ exons. A polyadenylation signal within an intron allows this single gene to encode two polypeptides. We hypothesize that altemative processing of a transcript is a basis for channeling metabolic intermediates into two different pathways.

Identification of the enzymatic reactions encoded by the purG and purI genes of Escherichia coli.

The chromosomal locations of the genes purG and purI on the Escherichia coli linkage map are the opposites of those of Salmonella typhimurium. By methods which permit the identification of lesions in any of the five early enzymes of the purine de novo pathway, the gene-enzyme relationships of the purG and purI loci have been reevaluated in these two organisms. The results demonstrate that the relative locations of the genes encoding the two enzymes (phosphoribosylformylglycinamidine synthetase and phosphoribosylaminoimidazole synthetase) are similar in the two organisms. The gene products have been correctly determined in S. typhimurium. The gene products currently listed for the loci in E. coli are incorrect. The E. coli purG locus is equivalent to the S. typhimurium purI locus, and the E. coli purI locus is equivalent to the S. typhimurium purG locus.

Effect of nitrous oxide-induced inactivation of vitamin B 12 on glycinamide ribonucleotide transformylase and 5-amino-4-imidazole carboxamide transformylase

Exposure to nitrous oxide (N 2O) in vivo is accompanied by oxidation of cob[I]-alanin to the inactive cob[III]alamin [1]. There is loss of methionine synthetase activity [2] and evidence of depressed supply of single carbon units at the formate level of oxidation [3,4,5]. We measured the effect of inactivation of B 12 on the folate-dependent transformylases concerned in purine synthesis. After 24 h exposure to N 2O there was a significant fall in glycinamide ribonucleotide transformylase (EC 2.1.2.2) and a significant increase in 5-amino-4-imidazole carboxamide transformylase (EC 2.1.2.3).

Polyglutamyl folate coenzymes and inhibitors of chicken liver glycinamide ribotide transformylase

Chicken liver glycinamide ribotide transformylase (5,10-methenyltetrahydrofolate:5'phosphoribosylglycinamide formyltransferase, EC 2.1.2.2), an enzyme of purine biosynthesis de novo, has greater specificity for its poly-γ-glutamyl folate coenzymes, 5,10-methenyltetrahydropteroyl(glutamate) n, where n = 3, 4, 5, 6 or 7, when compared to the monoglutamyi folate coenzyme. The relative specificity constants ( V/ K m) for the coenzymes 5,10-methenyltetrahydropteroyl(glutamate) n are 1.0, 1.6, 2.6, 2.4, 2.4, 4.9 and 1.5 for n=1, 2, 3, 4, 5, 6 and 7, respectively. Pteroylpoly-γ-(glutamate) n, where n=3, 4, 5, 6 or 7, are much better inhibitors of this enzyme when compared to the pteroylmonoglutamate. The concentration of inhibitor required for 50% inhibition was found to be 490, 120, 58, 28, 16, 14 and 12 μM for n=1, 2, 3, 4, 5, 6 and 7, respectively. Inhibitors with four or more glutamic acid residues gave grossly non-linear Dixon plots, in contrast to the linear plots obtained using inhibitors with three or less glutamic acid residues. The above findings make it feasible for the activity of glycinamide ribotide transformylase to be regulated by alteration in the length of the poly-γ-glutamyl chain of its folate coenzymes and of folate inhibitors.

Developmental changes in the folate-dependent enzymes of de novo purine biosynthesis in rat brain.

The activities of the two folate-dependent enzymes in the de novo purine biosynthetic pathway (e.g., glycinamide ribonucleotide transformylase and aminoimidazolecarboxamide ribonucleotide transformylase), have been evaluated as a function of age in crude extracts from rat brain, liver, kidney, and spleen. The activities of the enzymes in brain are similar to those found in liver and kidney. In all tissues the activity of both enzymes was higher during early development, more than nine times above adult levels. In the CNS the enzymatic activities are apparently related to the periods of increased nucleic acid synthesis, with different activities being found in different regions during development. Our findings lend strong support to the suggestion that folic acid-dependent metabolism plays an important role during early development of the brain.

ChemInform Abstract: SYNTHESIS OF 5,11‐METHENYLTETRAHYDROHOMOFOLATE AND ITS ANTIFOLATE ACTIVITY IN VITRO

AbstractDurch Cyclisierung der Tetrahydroverbindung (I) erhält man die im Titel genannte Verbindung (II), die in vitro 5,11‐Methenyl‐tetrahydrofolat‐Cyclohydrolase inhibiert.

Synthesis of 5,11-methenyltetrahydrohomofolate and its antifolate activity in vitro

The synthesis of 5,11-methenyltetrahydrohomofolate was accomplished by treatment of tetrahydrohomofolate (H4homofolate) with triethyl orthoformate in glacial acetic acid. This compound is a homofolate analogue of 5,10-methenyltetrahydrofolate which serves as one precursor to the 10-formyl one-carbon donor for the first transformylation in de novo purine biosynthesis, namely, the conversion of glycinamide ribonucleotide (GAR) to N-formylglycinamide ribonucleotide (FGAR), catalyzed by the enzyme glycinamide ribonucleotide transformylase (EC 2.1.2.2). The analogue proved to retard the rate of formation of formylglycinamide ribonucleotide apparently by inhibiting the rate of synthesis of 10-formyltetrahydrofolate, the actual cofactor for the transformylase enzyme, from 5,10-methenyltetrahydrofolate. Its inhibition of the enzyme, 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), was competitive against (+)-L-5,10-methenyltetrahydrofolate, with a Ki = 41 micro M. This derivative of homofolate may be responsible for its inhibition of purine biosynthesis in Sarcoma 180 cells.

L(-)-10-Formyltetrahydrofolate is the cofactor for glycinamide ribonucleotide transformylase from chicken liver

It is shown that L(-)-10-formyltetrahydrofolate serves as the cofactor for glycinamide ribonucleotide transformylase from chicken liver. The utilization of L(-)-10-formyl-H4folate was not previously recognized, because L-(+)-10-formyl-H4folate is an excellent competitive inhibitor of the enzyme, Ki = 0.75 +/- 0.07 microM, and historically the transformylase assay was carried out with a mixture of diastereomers. The results are discussed in relation to the utilization of L(+)-5,10-methenyl-H4folate.

On the cofactor specificity of glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase from chicken liver.

Tests of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and glycinamide ribonucleotide (GAR) transformylase cofactor specificity were conducted with 5-and/or 8-deazafolate analogues formylated at N-10. Several of these compounds were found to serve as cofactors for both the enzymes. The finding that 10-formyl-8-deazafolate can be used by AICAR transformylase eliminates those mechanisms requiring cyclization to a methenyl derivative prior to carbon unit transfer for this transformylase. Surprisingly, a similar analogue, 10-formyl-5,8-deazafolate, is very effective as a cofactor for GAR transformylase in the presence or absence of the trifunctional protein which is required for 5,10-methenyl-H4-folate activity with this transformylase. This finding suggests that the trifunctional protein modulates GAR transformylase cofactor specificity by supplying the active cofactor as the N10-formyl species, possibly through a transport process that avoids its dissociation into solution.

On the purification and mechanism of action of 5-aminoimidazole-4-carboxamide-ribonucleotide transformylase from chicken liver.

The transformylase from chicken liver catalyzing the formylation of 5-aminoimidazole-4-carboxamide ribonucleotide through the agency of 19-formyltetrahydrofolate has been purified to apparent homogeneity. Inosinicase activity copurifies. This transformylase is not further activated kinetically by the presence of the trifunctional protein in contrast to the glycinamide ribonucleotide transformylase. The enzyme exhibits a greater than 1000-fold preference for the naturally occurring 10-formyltetrahydrofolate cofactor and a sequential reaction pattern. A reinvestigation of the chemical structure of the formylated ribotide product employing 13C and 1H NMR indicated that the imidazole ring remained intact upon formylation, consistent with the originally proposed structure.

Characterization of the enzyme complex involving the folate-requiring enzymes of de novo purine biosynthesis.

Evidence is presented for a functional association of GAR TFase and the trifunctional protein within the protein complex consisting of GAR TFase, AICAR TFase, Ser HMase, and trifunctional protein. Resolution of the trifunctional protein from the remaining enzymes in the complex causes a loss of GAR TFase activity which is regained upon recombination. The minimum stoichiometry for GAR TFase reactivation is 3:1 GAR TFase--trifunctional protein. Determination by ultracentrifugation of the sedimentation coefficient as a function of protein concentration disclosed that the complex is in mobile equilibrium with the individual proteins. However, mixed GAR TFase--trifunctional protein species can be detected by trapping with cleavable bifunctional cross-linking reagents. Additional support for their interaction is found in the kinetic coupling of the trifunctional protein and GAR TFase activities that leads to a fourfold more efficient formation of formylglycinamide ribotide commencing with formate rather than with 5,10-methenyl-H4folate triglutamate.

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: isolation and characterization of a mutant deficient in the activity of phosphoribosylaminoimidazole synthetase.

A new purine-requiring mutant of Chinese hamster ovary cells (CHO-Kl) is described. This mutant, Ade-G, grows on aminoimidazole carboxamide, hypoxanthine, or adenine. It complements all eight of our other previously described Ade- mutants. Biochemical analysis of de novo purine synthesis in whole cells suggests that Ade-G is capable of the first four reactions of de novo purine biosynthesis and that it synthesizes and accumulates phosphoribosylformylglycinamidine (FGAM). Direct enzyme assay in cell-free extracts confirms that Ade-G is defective in phosphoribosylaminoimidazole synthetase activity and does not convert FGAM to phosphoribosylaminoimidazole (AIR), the next intermediate in the de novo biosynthetic pathway.

Repression and Derepression of Purine Biosynthesis in Mammalian Hepatoma Cells in Culture

Abstract The rates of biosynthesis of purines from glycine have been examined in an established line of rat hepatoma cells in continuous culture. The presence of 10-4 m adenine in the culture medium causes a prompt 4-fold to 5-fold decline in the rate of [14C]glycine incorporation into purines, and the capacity for de novo purine biosynthesis decays with a half-time of approximately 4 hours. Upon removal of adenine, the rate promptly increases, but the increase can be prevented by cycloheximide or actinomycin D. Neither cycloheximide nor actinomycin D prevents the decline in purine synthesis caused by adenine. Cycloheximide or actinomycin D alone causes a fall in the rate of purine formation, and the rate decays with a half-time of about 2 hours. The presence of 5-azacytidine, a pyrimidine analog which does not significantly inhibit RNA synthesis but is probably incorporated into RNA molecules, also causes the rate to decay with a similar half-time. The intracellular accumulation of α-N-formylglycinamide ribonucleotide (in the presence of 6.4 x 10-6 m azaserine in the culture medium) is repressed and derepressed by the addition and removal, respectively, of exogenous adenine. The kinetics of derepression and the inhibition by cycloheximide and 5-azacytidine of the rate of formylglycinamide ribonucleotide accumulation suggest that the regulation in the first third of the pathway is responsible for the control of the entire pathway by adenine. Under the conditions employed, the intracellular and extracellular concentrations of the precursor, glycine, do not change. The first three enzymes committed to de novo purine synthesis, phosphoribosylpyrophosphate glutamyl amidotransferase, glycinamide ribonucleotide synthetase, and glycinamide ribonucleotide transformylase, are not repressed or derepressed by the presence or absence of adenine in the culture medium. The identity of the repressible macromolecule(s) is unknown at present but it appears not to be specifically committed to purine biosynthesis.

Genetic Blocks and Unique Features in the Biosynthesis of 5′-Phosphoribosyl-N-formylglycinamide in Salmonella typhimurium

Purine-requiring mutants of Salmonella typhimurium have been examined for the ability to synthesize 5′-phosphoribosyl-N-formylglycinamide in a coupled reaction involving the first three enzymes of the biosynthetic pathway for purine nucleotides. Extracts prepared from mutants belonging to the distinct genetic classes, pur F and pur D, were inactive in this assay but were active when mixed together. Further analysis showed that pur F mutants were deficient in the first enzyme, 5-phosphoribosyl 1-pyrophosphate amidotransferase (ribosylamine-5-phosphate:pyrophosphate phosphoribosyltransferase, EC 2.4.2.14), and pur D mutants were apparently deficient in the second enzyme, phosphoribosylglycinamide synthetase (ribosylamine-5-phosphate:glycine ligase, EC 6.3.1.3). The third enzyme, phosphoribosylglycinamide formyltransferase (5′-phosphoribosyl-N-formylglycineamide:tetrahydrofolate 5,10-formyltransferase, EC 2.1.2.2), was present in all mutants, and a genetic deficiency for this enzyme has not been found. Attempts to limit the action of this enzyme by creating folate deficiencies were unsuccessful. This and other considerations suggest a uniqueness in the formyltransferase system different from the equivalent nonbacterial systems.

Biosynthesis of the purines.

GAR transformylase, which is responsible for the incorporation of formate carbon into what becomes position 8 of the purine ring, has been completely separated from 5-amino-4-imidazolecarboxamide ribotide transformylase (3). The latter enzyme is responsible for the incorporation of formate carbon into position 2 of the purine ring. The requirement for folic acid compounds has been demonstrated in both reactions of purine biosynthesis involving formate incorporation (4-7).