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PubMed1980

Pteroylpoly(gamma-glutamate) synthesis by Corynebacterium species. Purification and properties of folypoly(gamma-glutamate) synthetase.

Folylpolyglutamate synthetase was purified 7000-fold from extracts of Corynebacterium sp. The final preparation, which was greater than 95% pure, had a monomer molecular weight of 53,000. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to a variety of reduced pteroate and reduced pteroylmono-, di-, and triglutamate substrates with the concomitant production of ADP and Pi. Although the specificity for the folate substrate was wide, the pteroate and pteroylmonoglutamate substrates were utilized much more effectively than the polyglutamate derivatives. The most effective substrates were tetrahydropteroate, tetrahydrofolate, and 5,10-methylene-tretrahydrofolate. The most effective diglutamate substrate was 5,10-methylene-tetrahydropteroyldiglutamate. Addition of more than one glutamate moiety was only observed with tetrahydropteroate and 5,10-methylene-tetrahydropteroylmono- and diglutamates as substrates. The enzyme exhibited a preference for ATP as the nucleotide substrate. dATP was almost as effective while UTP and CTP were less effective substitutes. The specificity for L-glutamate appeared to be absolute. Enzyme activity was maximal at about pH 10 and exhibited an absolute requirement for a monovalent cation, of which K+ was the most effective. Preliminary studies suggest that the active form of the enzyme may be a dimer and that K+ may be required to effect dimerization.

Adenosine Triphosphate↗
PubMed1983

A structural role for dihydropteroyl hexaglutamate in the tail baseplate of various bacteriophages.

A novel non-metabolic role is proposed for dihydropteroyl hexaglutamate as a critical link binding together sub-structures of the tail of Escherichia coli bacteriophage T4. Six molecules of this folate compound have been found to be components of the complex tail baseplate of the phage particle. The baseplate is assembled using a total of at least 18 viral gene products in a series of reactions in which six wedge-like elements (each 0.7 X 10(6) daltons) bind symmetrically around a central tail plug (1.55 X 10(6) daltons) to form a flat hexagonal structure. It appears likely that the pteridine portion of the folate binds to a site on a viral-induced dihydrofolate reductase molecule, a wedge component, while the glutamate residues of the folate bind to a viral-induced thymidylate synthase molecule, a central plug component. Additionally, it appears that the folyl glutamate residues play a role in forming a flexible bond between the proximal end of the phage long tail fiber and the baseplate. Two bacteriophages attacking a quite different bacterial host, Pseudomonas syringae, have been isolated and partially characterized. Both phage strains have tail structures morphologically analogous to T4. Both were irreversibly inactivated by an enzyme which cleaves the gamma-glutamyl bonds of folyl polyglutamate. It appears that these Pseudomonas phage particles also contain a folyl poly-glutamate whose integrity is essential for their infectivity.

Escherichia coli↗
PubMed1989

Interaction of folylpolyglutamates with enzymes in one-carbon metabolism.

Of all the coenzymes, tetrahydrofolate exhibits the most structural diversity. The relationship of these structural forms to physiological function is under intense study by numerous research groups. In textbooks, tetrahydrofolate (tetrahydropteroylmonoglutamate) is shown as the coenzyme of one-carbon metabolism, but it has been known for several decades that the physiologically active forms of the coenzyme contain from 4 to 7 glutamyl residues linked by amide bonds through the gamma-carboxyl group. These glutamyl residues do not serve a direct function in transferring the one-carbon group. The tetrahydrofolylpolyglutamates were originally thought to be simply storage forms of the coenzyme, but studies now show that the polyglutamate chain of the coenzyme affects the transport properties of the coenzyme, alters the kinetic properties of many enzymes in one-carbon metabolism, and results in channeling of the coenzyme between several enzymes. In general, the dissociation constants of this group of enzymes for the tetrahydrofolylpolyglutamates are very low, in the 0.1 to 1 microM range. The concentration of the coenzyme in the cell appears to be similar to the concentration of folate-utilizing enzymes, suggesting that the concentration of unbound coenzyme in the cell may be very low. Several of the enzymes in one-carbon metabolism are either multifunctional proteins or multienzyme complexes. An active area of research is to determine if there is a functional relationship between these multifunctional enzymes and the polyglutamate portion of the coenzyme.

Carbon↗
PubMed1985

The effect of ethanol consumption on folate polyglutamate biosynthesis in the rat.

Contrary to previous studies, the folate polyglutamate chain length in the rat liver appears to be unaffected by ethanol ingestion for periods of 2-13 weeks. The appearance of short chain length folates after 13 weeks has been shown to arise as a result of increased in vitro folate polyglutamate breakdown during extraction due to a greater release of lysosomal conjugase in these animals.

Animals↗
PubMed1985

Determination of mouse liver 5-methyltetrahydrofolate concentration and polyglutamate forms.

The concentration and polyglutamate status of 5-methyltetrahydrofolate in mouse liver tissue extracts has been determined by enzymatic conversion to methylenetetrahydrofolate and subsequent entrapment of this cofactor form into a ternary complex with Lactobacillus casei thymidylate synthase and tritiated 5-fluorodeoxyuridylate. 5-Methyltetrahydrofolate was oxidized to methylenetetrahydrofolate using the reverse reaction of methylenetetrahydrofolate reductase with menadione as the ultimate electron acceptor. Reference 5-methyltetrahydrofolate could be quantitatively recovered from tissue extracts by this method. The polyglutamate status of enzymatically converted and complexed tissue 5-methyltetrahydrofolate was determined electrophoretically. Unlabeled 5-fluorodeoxyuridylate was used to remove endogenous methylenetetrahydrofolate prior to enzymatic oxidation of 5-methyltetrahydrofolate and subsequent electrophoretic analysis. In this manner, the 5-methyltetrahydrofolate polyglutamate pool alone could be labeled and visualized. There were no observable differences in the polyglutamate distribution of endogenous methylenetetrahydrofolate versus 5-methyltetrahydrofolate polyglutamates in extracts of normal mouse liver tissue.

Animals↗
PubMed1979

Folates of rat tissue. Bioassay of tissue folylpolyglutamates and a relationship of liver folypolyglutamates to nutritional folate sufficiency.

The folate content of young rat tissues extracted into boiling ascorbate was assayed by Lactobactillus casei both without and after treatment by a folate-free preparation of conjugase. The total folate content of various tissues was: liver, 8.9 microgram/g; kidney, 2.6; adrenal, 2.6; bone marrow, 2.4; spleen, 0.9; erythrocytes, 0.8; small intestinal mucosa, 0.7; small intestinal smooth muscle, 0.8; heart, 0.6; brain, 0.4, and skeletal muscle, 0.1 microgram/g tissue. For most tissues, with the exception of muscle and kidney, approximately 80% of the total folates assayed as longer chain length folylpolyglutamates. When liver folates were analyzed from rats fed folate-supplemented, control and folate-deficient diets, a relationship was found between folate nutrition and distribution of folylpolyglutamates. The proportion of total folates in the form of longer chain length folylpolyglutamates was greatest in the livers of folate-deficient rats and least in the livers of folate-supplemented rats.

Animals↗
PubMed1980

Interactions of pig liver methylenetetrahydrofolate reductase with methylenetetrahydropteroylpolyglutamate substrates and with dihydropteroylpolyglutamate inhibitors.

Dihydrofolate and dihydropteroylpolyglutamates inhibit pig liver methylenetetrahydrofolate reductase. In all cases the inhibition is linearly competitive with respect to methylenetetrahydrofolate. The Ki values decrease with each additional glutamyl residue from one to six, from a value of 6.5 microM for dihydrofolate to 0.013 microM for dihydropteroylhexaglutamate. Dihydropteroylheptaglutamate has a Ki of 0.065 microM. These data indicate a free energy of binding of approximately 0.75 kcal/mol for each of the five terminal glutamyl residues in dihydropteroylhexaglutamate. Methylenetetrahydropteroylpolyglutamates are substrates for the enzyme, and the increased free energy of binding is reflected in increased values for Vmax/Km with polyglutamate substrates. Vmax is increased 1.76-fold on going from the mono- to the diglutamate substrate; additional glutamyl residues lead to decreases in Km values for methylenetetrahydropteroylpolyglutamates. Our results suggest that the in vivo activity of methylenetetrahydrofolate reductase may also be sensitive to fluctuations in the ratio of methylenetetrahydropteroylpolyglutamates to dihydropteroylpolyglutamates and that this ratio may be important in determining the relative fluxes of methylenetetrahydropteroylpolyglutamates into the pathways leading to thymidylate biosynthesis and methionine regeneration.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗
PubMed1979

Folylpoly-gamma-glutamates as cosubstrates of 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase.

N10-Formyltetrahydropteroylpoly-gamma-glutamates (N10-formyl-H4PteGlun) having n = 1, 3, 4, 5, 6, and 7 glutamyl residues have been tested as cosubstrates of the purine biosynthesis enzyme 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase (AICAR transformylase) of chicken liver. The cosubstrates were synthesized by solid-phase synthesis, reduced catalytically, and formylated; a purified enzyme preparation was used and assayed spectrophotometrically following the deltaOD at 298 nm resulting from conversion of the formylated folate to the free tetrahydro form. Km values and Vm values determined at saturating concentrations of AICAR and at 25 and 150 mM KC1 were used to calculate the relative specificity constants Vm/Km for the N10-formyl-H4PteGlun. At physiologic [K+] (150 mM) they were 1.0, 52, 250, 93, 120, and 59 and at the lower (25 mM) [K+] the relative specificity constants were 1.0, 64, 78,34, 48, and 37 for n = 1, 3, 4, 5, 6, and 7, respectively. The poly-gamma-glutamates are clearly the preferred cosubstrates, particularly when tested at physiologic [K+]. The maximal relative specificity constant observed with N10-formyl-H4PteGlu4 supports the hypothesis that regulation of certain pathways of one-carbon metabolism may operate via alterations of the poly-gamma-glutamyl chain length. No inhibition by the unnatural (d) isomers of the N10-formyl-H4PteGlun was observed.

Acyltransferases↗
PubMed1980

5-fluorouracil-methotrexate synergy: enhancement of 5-fluorodeoxyridylate binding to thymidylate synthase by dihydropteroylpolyglutamates.

Ternary complex formation of thymidylate synthase (5,10-methylenetetrahydrofolated:dUMP C-methyltransferase, EC 2.1.1.45), 5-fluorodeoxyuridylate (FdUMP), and poly(gamma-glutamyl) conjugates of pteroate and methotrexate (MTX) has been examined as a basis for the sequence-dependent synergism of the 5-fluorouracil-MTX combination in inhibiting viability of L1210 murine tumor cells. A 1.4-log (25-fold) increase in the inhibition of soft agar colony formation was observed when MTX preceded 5-fluorouracil as compared to the reverse sequence. L1210 cells converted 39% of the total intracellular MTX into MTX poly(gamma-glutamate)s within 4 hr of exposure to 1 microM MTX. MTX and MTX(gamma-glutamate) formed reversible ternary complexes with FdUMP on one site of thymidylate synthase, whereas with 7,8-dihydropteroylpentaglutamate and I-5,10-methylenetetrahydropteroylpentaglutamate stoichiometric binding of FdUMP to two sites on thymidylate synthase was observed. The dissociation constants for FdUMP in the ternary complexes formed in the presence of MTX, MTX(gamma-glutamate), 7,8-dihydropteroylpentaglutamate, and I-5-10-methylenetetrahydropteroylpentaglutamate were estimated to be 370, 27, < 10, and < 10 nM, respectively, by equilibrium dialysis. We propose that the sequence-dependent effect of MTX plus 5-fluorouracil on L1210 cell viability results from MTX and MTX polyglutamate inhibition of dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) and consequently a trapping of intracellular folates as dihydropteroylpolyglutamates, which increase the extent of FdUMP binding to thymidylate synthase.

Animals↗
PubMed1981

Dual functions of bacteriophage T4D gene 28 product: structural component of the viral tail baseplate central plug and cleavage enzyme for folyl polyglutamates. II. Folate metabolism and polyglutamate cleavage activity of uninfected and infected Escherichia coli cells and bacteriophage.

We investigated the role of the T4D bacteriophage gene 28 product in folate metabolism in infected Escherichia coli cells by using antifolate drugs and a newly devised assay for folyl polyglutamate cleavage activity. Preincubation of host E. coli cells with various sulfa drugs inhibited phage production by decreasing the burst size when the phage particles produced an altered gene 28 product (i.e., after infection under permissive conditions with T4D 28(ts) or T4D am28). In addition, we found that another folate analog, pyrimethamine, also inhibited T4D 28(ts) production and T4D 28am production, but this analog did not inhibit wild-type T4D production. A temperature-resistant revertant of T4D 28(ts) was not sensitive to either sulfa drugs or pyrimethamine. We developed an assay to measure the enzymatic cleavage of folyl polyglutamates. The high-molecular-weight folyl polyglutamate substrate was isolated from E. coli B cells infected with T4D am28 in the presence of labeled glutamic acid and was characterized as a folate compound containing 12 to 14 labeled glutamate residues. Extracts of uninfected bacteria liberated glutamate residues from this substrate with a pH optimum of 8.4 to 8.5. Extracts of bacteriophage T4D-infected E. coli B cells exhibited an additional new folyl polyglutamate cleavage activity with a pH optimum of about 6.4 to 6.5, which was clearly distinguished from the preexisting activity in the uninfected host cells. This new activity was induced in E. coli B cells by infection with wild-type T4D and T4D amber mutants 29(-), 26(-), 27(-), 51(-), and 10(-), but it was not induced under nonpermissive conditions by T4D am28 or by T4D 28(ts). Mutations in gene 28 affected the properties of the induced cleavage enzyme. Wild-type T4D-induced cleavage activity was not inhibited by pyrimethamine, whereas the T4D 28(ts) activity induced at a permissive temperature was inhibited by this folate analog. Folyl polyglutamate cleavage activity characteristic of the activity induced in host cells by wild-type T4D or by T4D gene 28 mutants was also found in highly purified preparations of these phage ghost particles. The T4D-induced cleavage activity could be inhibited by antiserum prepared against highly purified phage baseplates. We concluded that T4D infection induced the formation of a new folyl polyglutamate cleavage enzyme and that this enzyme was coded for by T4D gene 28. Furthermore, since this gene product was a baseplate tail plug component which had both its antigenic sites and its catalytic sites exposed on the phage particle, it was apparent that this enzyme formed part of the distal surface of the phage baseplate central tail plug.

Carboxypeptidases↗