7,8-Dihydropteroyl oligo-γ-L-glutamates. Synthesis and kinetic studies with purified dihydrofolate reductase from mammalian sources

Coward, James K.; Parameswaran, K. N.; Cashmore, Arlene R.; Bertino, Joseph R.

doi:10.1021/bi00716a013

Cited by 92 publications

(31 citation statements)

References 25 publications

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“…This report describes the 2500-fold enhanced capacity of MTX polyglutamate to inhibit 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase [5-amino-4-imidazolecarboxamide ribotide (AICAR) transformylase, EC 2.1.2.3; AICAR TFase], a folate-requiring enzyme that catalyzes the reaction: 10-formyl-tetrahydrofolate (10-formyl-H4PteGlu) + AICAR, yielding 5'-phosphoribosyl-5-formamido-4-imidazole-carboxamide (formyl-AICAR), an intermediate in the de novo purine biosynthetic pathway, and tetrahydrofolic acid (H4PteGlu). We also report that H2PteGlu5, which increases in the cell following inhibition of H2PteGlu reductase (11) (Glu-Glu), and H2PteGlu were purchased from Sigma, and H2PteGlu5 was reduced from PteGlu5 as described (13,14) and purified by recrystallization. Affi-Gel Blue was purchased from Bio-Rad.…”

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confidence: 99%

“…This report describes the 2500-fold enhanced capacity of MTX polyglutamate to inhibit 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase [5-amino-4- (12). AICAR, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, 'y-L-glutamyl-L-glutamic acid (Glu-Glu), and H2PteGlu were purchased from Sigma, and H2PteGlu5 was reduced from PteGlu5 as described (13,14) and purified by recrystallization. Affi-Gel Blue was purchased from Bio-Rad.…”

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confidence: 99%

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Inhibition of phosphoribosylaminoimidazolecarboxamide transformylase by methotrexate and dihydrofolic acid polyglutamates.

Allegra¹,

Drake²,

Jolivet³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

258

137

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We report the enhanced inhibitory potency of methotrexate (MTX) polyglutamates and dihydrofolate pentaglutamate on the catalytic activity of phosphoribosylaminoimidazolecarboxamide (AICAR) transformylase purified from MCF-7 human breast cancer cells. In the present work, MTX (4-amino-10-methylpteroylglutamic acid) and dihydrofolate, both monoglutamates, were found to be weak competitive inhibitors of AICAR transformylase with Kis of 143 and 63 ,uM, respectively, and their inhibitory capacity was largely unaffected by the glutamated state of the folate cosubstrate. In contrast, MTX polyglutamates were found to be potent competitive inhibitors, with an =10-fold increase in inhibitory potency with the addition of each glutamate group up to four (i.e., the pentaglutamate derivative). MTX (2), and the polyglutamated forms of MTX have been detected in a number of neoplastic and, to a lesser extent, normal tissues following MTX exposure (3-9). While retaining a potent inhibitory effect on H2PteGlu reductase, the polyglutamated forms of MTX differ from parent MTX in that they possess a more prolonged intracellular half-life resulting from a slower efflux rate from cells (10). The selective retention of the MTX polyglutamates results in prolonged antimetabolic effects after the removal of extracellular drug.We have investigated the possibility that polyglutamation of MTX may increase its ability to inhibit other folatedependent enzymes, particularly those that have a higher affinity for polyglutamated folate substrates. This report describes the 2500-fold enhanced capacity of MTX polyglutamate to inhibit 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase [5-amino-4-imidazolecarboxamide ribotide (AICAR) transformylase, EC 2.1.2.3; AICAR TFase], a folate-requiring enzyme that catalyzes the reaction: 10-formyl-tetrahydrofolate (10-formyl-H4PteGlu) + AICAR, yielding 5'-phosphoribosyl-5-formamido-4-imidazole-carboxamide (formyl-AICAR), an intermediate in the de novo purine biosynthetic pathway, and tetrahydrofolic acid (H4PteGlu). We also report that H2PteGlu5, which increases in the cell following inhibition of H2PteGlu reductase (11) (Glu-Glu), and H2PteGlu were purchased from Sigma, and H2PteGlu5 was reduced from PteGlu5 as described (13,14) and purified by recrystallization. Affi-Gel Blue was purchased from Bio-Rad. All other chemicals were of reagent grade and purchased from Sigma.Preparation of Reduced Folates. Pure, biologically active l-L-10-formyl-H4PteGlu and -Glu5 were prepared by enzymatic reduction of H2PteGlu or -Glu5 to H4PteGlu or -Glu5 (15). For the enzymatic reduction, H2PteGlu or -Glu5 (50 mg) and NADPH (125 mg) in 20 ml 0.05 M Tris HCl buffer (pH 7.4) were incubated at 370C with partially purified Lactobacterium casei H2PteGlu reductase (New England Enzyme Center, Boston). The reaction was followed spectrophotometrically at 340 nm until no additional NADPH was metabolized. The H4PteGlu and -Glu5 thus formed were purified by elution from a DEAE-cel...

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“…This report describes the 2500-fold enhanced capacity of MTX polyglutamate to inhibit 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase [5-amino-4- (12). AICAR, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, 'y-L-glutamyl-L-glutamic acid (Glu-Glu), and H2PteGlu were purchased from Sigma, and H2PteGlu5 was reduced from PteGlu5 as described (13,14) and purified by recrystallization. Affi-Gel Blue was purchased from Bio-Rad.…”

mentioning

confidence: 99%

Inhibition of phosphoribosylaminoimidazolecarboxamide transformylase by methotrexate and dihydrofolic acid polyglutamates.

Allegra¹,

Drake²,

Jolivet³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

258

137

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confidence: 99%

“…The rate-limiting step in the utilization of pteroylpolyglutamates by this organism is transport into the cell, while intracellular metabolism, and not transport, is rate-limiting with pteroylmonoglutamates. In vitro studies have also shown that pteroylpolyglutamates are often better substrates than the corresponding monoglutamates for folate-requiring enzymes (Burton & Metzenberg, 1975;Cheng, Shane & Stokstad, 1975;Coward et al, 1974 Coward et al, , 1975Curthoys & Rabinowitz, 1972;Kisliuk, Gaumont & Baugh, 1974;Powers & Snell, 1976;Salem, Pattison & Foster, 1972;Whitfield, Steers & Weissbach, 1970).To investigate which step in pteroylmonoglutamate metabolism might be rate-limiting, the metabolism of 5-[Me-14C]methyl-H,PteGlu and 5-methyl-H,[3H]PteGlu (for nomenclature, see Methods) were compared in order to assess the turnover of intracellular folate via various metabolic pathways. Although two mechanisms have been described for 5-methyl-H4PteGlu formation in bacteria involving the reduction of 5, I o-methylene-H,PteGlu or the direct transfer of a methyl group from trimethylsulphonium to H,PteGlu (Wagner et al, 1967), the only known route for the metabolism of methyltetrahydrofolates is via methionine synthesis (Taylor & Weissbach, 1973) but attempts to detect methionine synthetase activity in L. casei have proved unsuccessful (Galivan, 1971 ;Kisliuk, 1971).…”

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Metabolism of 5-Methyltetrahydrofolate by Lactobacittus casei

Shane¹,

Stokstad²

1977

Journal of General Microbiology

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The metabolism of 5-[Me-14C]methyltetrahydrofolate in Lactobacillus casei proceeded oxidatively with incorporation of label into purine and thymidylate derivatives. No labelled methionine was formed.(1)-5-Methyltetrahydrofolate, the natural isomer, was not a substrate for the L. casei folylpoly-y-glutamate synthetase although the unnatural (d)-isomer was slowly metabolized to the diglutamate form. I N T R O D U C T I O NWe have previously shown that pteroylpolyglutamates, once transported, are better growth promoters of Lactobacillus casei than pteroylmonoglutamates (Shane & Stokstad, I 976). The rate-limiting step in the utilization of pteroylpolyglutamates by this organism is transport into the cell, while intracellular metabolism, and not transport, is rate-limiting with pteroylmonoglutamates. In vitro studies have also shown that pteroylpolyglutamates are often better substrates than the corresponding monoglutamates for folate-requiring enzymes (Burton & Metzenberg, 1975;Cheng, Shane & Stokstad, 1975;Coward et al., 1974 Coward et al., , 1975Curthoys & Rabinowitz, 1972;Kisliuk, Gaumont & Baugh, 1974;Powers & Snell, 1976;Salem, Pattison & Foster, 1972;Whitfield, Steers & Weissbach, 1970).To investigate which step in pteroylmonoglutamate metabolism might be rate-limiting, the metabolism of 5-[Me-14C]methyl-H,PteGlu and 5-methyl-H,[3H]PteGlu (for nomenclature, see Methods) were compared in order to assess the turnover of intracellular folate via various metabolic pathways. Although two mechanisms have been described for 5-methyl-H4PteGlu formation in bacteria involving the reduction of 5, I o-methylene-H,PteGlu or the direct transfer of a methyl group from trimethylsulphonium to H,PteGlu (Wagner et al., 1967), the only known route for the metabolism of methyltetrahydrofolates is via methionine synthesis (Taylor & Weissbach, 1973) but attempts to detect methionine synthetase activity in L. casei have proved unsuccessful (Galivan, 1971 ;Kisliuk, 1971). METHODSNomenclature. The abbreviations used are : PteGlu, pteroylglutamic acid, folic acid; PteGlu,, pteroylmonoto pteroyloligo-y-L-glutamic acid, n indicating the number of glutamic; acid residues; H,PteGlu,, 5,6,7,8-tetrahydropteroylmono-to ~,6,~,8-tetrahydropteroyloligo-y-~-glutamic acid. The symbols ( I ) and (d) are used to denote the natural and unnatural diastereoisomers of H,PteGlu,, respectively, due to the asymmetric centre at the C-6 position, and do not indicate optical activity.

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“…Polyglutamate is a suitable substrate for the majority of folate-dependent reactions [20], so one would expect decreased intracellular folate activity in the presence of alcohol. It may be that this is a phenomenon unique to the tissues that we have examined, and further studies are required on this point.…”

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The Toxic Effects of Alcohol on Folate Metabolism

Steinberg

Campbell

Hillman

1980

Clinical Toxicology

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Alcoholism is associated with a number of changes in cellular metabolism which, in time, may lead to organ damage. In the case of rapidly proliferating tissues, alcohol-induced defects i n folate metabolism can compromise cell replication. For example, in the hematopoietic system, megaloblastic anemia, leukopenia, and thrombocytopenia are common manifestations p-31. The toxic effect of alcohol on folate homeostasis is the result of a combination of factors. Dietary deprivation results from the marginal nutritional status that frequently accompanies alcoholism [4]. Intestinal malabsorption of folate acid can also occur in the setting of alcohol-related protein malnutrition [5]. However, while these effects contribute to folate deficiency in the chronic state, they cannot explain the alterations in folate metabolism which have been demonstrated with acute alcohol ingestion [ 6 ] .Sullivan and Herbert first studied the toxic effect of alcohol in patients with alcohol-induced megaloblastic erythropoiesis undergoing folate therapy [ 11. Alcohol appeared to "block" folate activity. Physiologic doses of pteroylglutamic acid which were sufficient to correct folate deficiency in control subjects were ineffective in patients main-*To whom reprint requests should be addressed. 407

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7,8-Dihydropteroyl oligo-γ-L-glutamates. Synthesis and kinetic studies with purified dihydrofolate reductase from mammalian sources

Cited by 92 publications

References 25 publications

Inhibition of phosphoribosylaminoimidazolecarboxamide transformylase by methotrexate and dihydrofolic acid polyglutamates.

Inhibition of phosphoribosylaminoimidazolecarboxamide transformylase by methotrexate and dihydrofolic acid polyglutamates.

Metabolism of 5-Methyltetrahydrofolate by Lactobacittus casei

The Toxic Effects of Alcohol on Folate Metabolism

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