Interaction of tetrahydropteroylpolyglutamates with two enzymes from mitochondria

Strong, William B.; Cook, Robert; Schirch, Verne

doi:10.1021/bi00427a016

Cited by 15 publications

(16 citation statements)

References 31 publications

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“…The reverse reaction has been demonstrated to proceed in coupled reactions in vitro (Strong & Schirch, 1989) and in vivo in yeast (McKenzie & Jones, 1977;Pasternack et al, 1992Pasternack et al, , 1994, demonstrating the incorporation of formate into the methyleneH4folate pool. The reverse reaction is clearly metabolically important.…”

Section: Discussionmentioning

confidence: 99%

Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase

Pelletier

MacKenzie²

1995

Biochemistry

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The bifunctional dehydrogenase/cyclohydrolase domain of the human NADP-dependent trifunctional methyleneH4folate dehydrogenase/methenylH4folate cyclohydrolase/formylH4folate synthetase (H4folate = tetrahydrofolate) catalyzes two sequential reactions involved in the interconversion of H4folate derivatives. We have established by equilibrium dialysis that a single H4folate-binding site exists per monomer of the dimeric domain and that the presence of nucleotides has two unexpected effects on H4folate substrate binding. Nucleotides containing a 5'-phosphate cause positive cooperativity in the binding of methyleneH4folate but not of 10-formylH4folate, and NADP increases the affinity for 10-formylH4folate by a factor of 25. The results indicate that dinucleotide preferentially binds before 10-formylH4folate in the reverse cyclohydrolase reaction, and this mechanism increases the efficiency of conversion of 10-formylH4folate to methyleneH4folate. We report new kinetic data that are also consistent with a steady-state random mechanism for this enzyme. To assess whether the enzyme functions at equilibrium in vivo, we determined the overall chemical equilibrium constant of Keq = 16 for ([10- formylH4folate][NADPH])/([methyleneH4folate][NADP]). Using this value and reported ratios of free dinucleotides and folate derivatives in vivo, we estimate that the cytosolic dehydrogenase/cyclohydrolase reactions exist near the equilibrium position. However, the NAD-dependent dehydrogenase/cyclohydrolase reactions in mitochondria are far from equilibrium and are poised toward 10-formylH4folate synthesis. The results of the binding and kinetic studies indicate that the bifunctional nature of the methyleneH4folate dehydrogenase/methenylH4folate cyclohdrolase domain is designed to optimize the overall reverse reactions in vivo.

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Section: Discussionmentioning

confidence: 99%

Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase

Pelletier

MacKenzie²

1995

Biochemistry

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“…Determination of Dissociation Constant for Reduced Folates. The dissociation constants for THF and N 5 -CHO-THF of zmSHMT(DelSig) were measured by the formation of quinonoid complex in the presence of saturated glycine and reduced folates ranging from 2.5 to 54 M (Strong et al, 1989;Chang et al, 2006). Results were analyzed with Scatchard plots and doublereciprocal plots, yielding K d and stoichiometry of bound folates.…”

Section: Methodsmentioning

confidence: 99%

Serine Hydroxymethyltransferase Isoforms Are Differentially Inhibited by Leucovorin: Characterization and Comparison of Recombinant Zebrafish Serine Hydroxymethyltransferases

et al. 2007

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ABSTRACT:Serine hydroxymethyltransferase (SHMT) provides activated onecarbon units required for the biosynthesis of nucleotides, protein, and methyl group by converting serine and tetrahydrofolate to glycine and N 5 ,N 10 -methylenetetrahydrofolate. It is postulated that SHMT activity is associated with the development of methotrexate resistance and the in vivo activity of SHMT is regulated by the binding of N 5 -CHO-THF, the rescue agent in high-dose methotrexate chemotherapy. The aim of this study is to advance our understanding of the folate-mediated one-carbon metabolism in zebrafish by characterizing zebrafish mitochondrial SHMT. The cDNA encoding zebrafish mitochondrial SHMT was cloned, overexpressed in Escherichia coli, and purified with a three-step purification protocol. Similarities in structural, physical, and kinetic properties were revealed between the recombinant zebrafish mitochondrial SHMT and its mammalian orthologs. Surprisingly, leucovorin significantly inhibits the aldol cleavage of serine catalyzed by zebrafish cytosolic SHMT but inhibits to a lesser extent the reaction catalyzed by the mitochondrial isozyme. This is, to our knowledge, the first report on zebrafish mitochondrial folate enzyme as well as the differential inhibition of leucovorin on these two SHMT isoforms. Western blot analysis revealed tissue-specific distribution with the highest enrichment present in liver for both cytosolic and mitochondrial SHMTs. Intracellular localization was confirmed by confocal microscopy for both mitochondrial and cytosolic SHMTs. Unexpectedly, the cytosolic isoform was observed in both nucleus and cytosol. Together with the previous report on zebrafish cytosolic SHMT, we suggest that zSHMTs can be used in in vitro assays for folate-related investigation and antifolate drug discovery.Folates carry the chemically activated single carbons at N 5 and/or N 10 positions and are required for the biosynthesis and metabolism of nucleic acid, protein, amino acid, methyl group, neurotransmitter, and vitamins. Its vital role in nucleotide biogenesis has led to the development of many anticancer drugs targeting folate-requiring enzymes. Among them, methotrexate (MTX) is one of the most widely used anticancer agents to date. It blocks de novo nucleotide synthesis by depleting reduced tetrahydrofolates mainly through inhibition of dihydrofolate reductase (DHFR) and thymidylate synthase (Fig. 1, enzymes 2 and 3, respectively). However, resistance to MTX often emerges and becomes the major impediment to its curative potential.To overcome this obstacle and prevent MTX-associated toxicity, high-dose MTX combined with leucovorin rescue is administered and has become an important regimen in the treatment of a variety of cancers (Frei et al., 1980). Despite these preventive measures, MTXinduced resistance and toxicity continue to occur, although infrequently. Mechanisms including elevated DHFR, decreased thymidylate synthase, impaired folate/antifolate transportation, and decreased polyglutamylation on MTX have been pr...

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“…Affinity of the Recombinant DNcSHMT Protein for 5-FormylTHFThe affinity of recombinant DNcSHMT protein for 5-formylTHF was determined by a previously described competitive binding assay (18).…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant DNcSHMT was added (to a final concentration of 25 M), and the spectrum was recorded. To quantify the affinity of DNcSHMT for 5-formylTHF, the loss of absorbance at 502 nm was recorded as a function of DNcSHMT added to the cuvette as described previously (18).…”

Section: Methodsmentioning

confidence: 99%

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Pyridoxal Phosphate Inhibits Dynamic Subunit Interchange among Serine Hydroxymethyltransferase Tetramers

Zanetti

2003

Journal of Biological Chemistry

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Cytoplasmic serine hydroxymethyltransferase (cSHMT) is a tetrameric, pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. The enzyme has four active sites and is best described as a dimer of obligate dimers. Each monomeric subunit within the obligate dimer contributes catalytically important amino acid residues to both active sites. To investigate the interchange of subunits among cSHMT tetramers, a dominant-negative human cSHMT enzyme (DNcSHMT) was engineered by making three amino acid substitutions: K257Q, Y82A, and Y83F. Purified recombinant DNcSHMT protein was catalytically inactive and did not bind 5-formyltetrahydrofolate. Coexpression of the cSHMT and DNcSHMT proteins in bacteria resulted in the formation of heterotetramers with a cSHMT/DNcSHMT subunit ratio of 1. Characterization of the cSHMT/DNcSHMT heterotetramers indicates that DNcSHMT and cSHMT monomers randomly associate to form tetramers and that cSHMT/ DNcSHMT obligate dimers are catalytically inactive. Incubation of recombinant cSHMT protein with recombinant DNcSHMT protein did not result in the formation of hetero-oligomers, indicating that cSHMT subunits do not exchange once the tetramer is assembled. However, removal of the active site PLP cofactor does permit exchange of obligate dimers among preformed cSHMT and DNcSHMT tetramers, and the formation of heterotetramers from cSHMT and DNcSHMT homodimers does not affect the activity of the cSHMT homodimers. The results of these studies demonstrate that PLP inhibits dimer exchange among cSHMT tetramers and suggests that cellular PLP concentrations may influence the stability of cSHMT protein in vivo.Serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) 1 is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate (THF) to glycine and methylenetetrahydrofolate (1, 2). This reaction is a primary source of single carbons that are required for cytoplasmic one-carbon metabolism. SHMT is present in the cytoplasm (cSHMT) and mitochondria of eukaryotic cells, and different genes encode the two SHMT proteins (3-5). Both SHMT isozymes are sources of one-carbon units for cytoplasmic one-carbon metabolism (1, 3). In the cytoplasm, folate-activated one-carbon units are required for the de novo synthesis of purines and thymidylate and for the remethylation of homocysteine to methionine (1, 6). Mitochondrial one-carbon metabolism is necessary for the conversion of serine to formate (7), and the mitochondrial SHMT enzyme catalyzes the first step in this pathway by converting serine and THF to glycine and methylenetetrahydrofolate (6, 7). Formate enters the cytoplasm, where it is incorporated into the folate one-carbon pool (1, 3, 4). The mitochondrial SHMT gene is expressed at similar levels in most mammalian tissues, whereas the cSHMT gene exhibits a dynamic range of tissue-specific expression (5).Mammalian SHMT enzymes are 55-kDa homotetram...

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Interaction of tetrahydropteroylpolyglutamates with two enzymes from mitochondria

Cited by 15 publications

References 31 publications

Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase

Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase

Serine Hydroxymethyltransferase Isoforms Are Differentially Inhibited by Leucovorin: Characterization and Comparison of Recombinant Zebrafish Serine Hydroxymethyltransferases

Pyridoxal Phosphate Inhibits Dynamic Subunit Interchange among Serine Hydroxymethyltransferase Tetramers

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