Pyridoxal phosphate inhibits dynamic subunit interchange among serine hydroxymethyltransferase tetramers

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.     

Structure determination and biochemical studies on Bacillus stearothermophilus E53Q serine hydroxymethyltransferase and its complexes provide insights on function and enzyme memory

Serine hydroxymethyltransferase (SHMT) belongs to the alpha-family of pyridoxal 5'-phosphate-dependent enzymes and catalyzes the reversible conversion of L-Ser and tetrahydrofolate to Gly and 5,10-methylene tetrahydrofolate. 5,10-Methylene tetrahydrofolate serves as a source of one-carbon fragment in many biological processes. SHMT also catalyzes the tetrahydrofolate-independent conversion of L-allo-Thr to Gly and acetaldehyde. The crystal structure of Bacillus stearothermophilus SHMT (bsSHMT) suggested that E53 interacts with the substrate, L-Ser and tetrahydrofolate. To elucidate the role of E53, it was mutated to Q and structural and biochemical studies were carried out with the mutant enzyme. The internal aldimine structure of E53QbsSHMT was similar to that of the wild-type enzyme, except for significant changes at Q53, Y60 and Y61. The carboxyl of Gly and side chain of L-Ser were in two conformations in the respective external aldimine structures. The mutant enzyme was completely inactive for tetrahydrofolate-dependent cleavage of L-Ser, whereas there was a 1.5-fold increase in the rate of tetrahydrofolate-independent reaction with L-allo-Thr. The results obtained from these studies suggest that E53 plays an essential role in tetrahydrofolate/5-formyl tetrahydrofolate binding and in the proper positioning of Cbeta of L-Ser for direct attack by N5 of tetrahydrofolate. Most interestingly, the structure of the complex obtained by cocrystallization of E53QbsSHMT with Gly and 5-formyl tetrahydrofolate revealed the gem-diamine form of pyridoxal 5'-phosphate bound to Gly and active site Lys. However, density for 5-formyl tetrahydrofolate was not observed. Gly carboxylate was in a single conformation, whereas pyridoxal 5'-phosphate had two distinct conformations. The differences between the structures of this complex and Gly external aldimine suggest that the changes induced by initial binding of 5-formyl tetrahydrofolate are retained even though 5-formyl tetrahydrofolate is absent in the final structure. Spectral studies carried out with this mutant enzyme also suggest that 5-formyl tetrahydrofolate binds to the E53QbsSHMT-Gly complex forming a quinonoid intermediate and falls off within 4 h of dialysis, leaving behind the mutant enzyme in the gem-diamine form. This is the first report to provide direct evidence for enzyme memory based on the crystal structure of enzyme complexes.     

Crystal structure of binary and ternary complexes of serine hydroxymethyltransferase from Bacillus stearothermophilus: insights into the catalytic mechanism

Serine hydroxymethyltransferase (SHMT), a member of the alpha-class of pyridoxal phosphate-dependent enzymes, catalyzes the reversible conversion of serine to glycine and tetrahydrofolate to 5,10-methylene tetrahydrofolate. We present here the crystal structures of the native enzyme and its complexes with serine, glycine, glycine, and 5-formyl tetrahydrofolate (FTHF) from Bacillus stearothermophilus. The first structure of the serine-bound form of SHMT allows identification of residues involved in serine binding and catalysis. The SHMT-serine complex does not show any significant conformational change compared with the native enzyme, contrary to that expected for a conversion from an "open" to "closed" form of the enzyme. However, the ternary complex with FTHF and glycine shows the reported conformational changes. In contrast to the Escherichia coli enzyme, this complex shows asymmetric binding of the FTHF to the two monomers within the dimer in a way similar to the murine SHMT. Comparison of the ternary complex with the native enzyme reveals the structural basis for the conformational change and asymmetric binding of FTHF. The four structures presented here correspond to the various reaction intermediates of the catalytic pathway and provide evidence for a direct displacement mechanism for the hydroxymethyl transfer rather than a retroaldol cleavage.     

The glycine-rich region of Escherichia coli D-serine dehydratase. Altered interactions with pyridoxal 5'-phosphate produced by substitution of aspartic acid for glycine

Replacement of glycine by aspartic acid at either of two sites in a conserved, glycine-rich region inactivates the pyridoxal 5'-phosphate-dependent enzyme D-serine dehydratase (DSD) from Escherichia coli. To investigate why aspartic acid at position 279 or 281 causes a loss of activity, we measured the affinity of the G----D variants for pyridoxal 5'-phosphate and a cofactor:substrate analog complex and compared the UV, CD, and fluorescence properties of wild-type D-serine dehydratase and the inactive variants. The two G----D variants DSD(G279D) and DSD (G281D) displayed marked differences from wild-type D-serine dehydratase and from each other with respect to their affinity for pyridoxal 5'-phosphate and for a pyridoxal 5'-phosphate:glycine Schiff base. Compared to the wild-type enzyme, the cofactor affinity of DSD(G279D) and DSD(G281D) was decreased 225- and 50-fold, respectively, and the ability to retain a cofactor:glycine complex was decreased 765- and 1970-fold. The spectral properties of the inactive variants suggest that they form a Schiff base linkage with pyridoxal 5'-phosphate but do not hold the cofactor in a catalytically competent orientation. Moreover, the amount of cofactor aldamine in equilibrium with cofactor Schiff base is increased in DSD(G279D) and DSD(G281D) relative to that in wild-type DSD. Collectively, our findings indicate that introduction of a carboxymethyl side chain at G-279 or G-281 directly or indirectly disrupts catalytically essential protein-cofactor and protein-substrate interactions and thereby prevents processing of the enzyme bound cofactor:substrate complex. The conserved glycine-rich region is thus either an integral part of the D-serine dehydratase active site or conformationally linked to it.     

Properties of human and rabbit cytosolic serine hydroxymethyltransferase are changed by single nucleotide polymorphic mutations

Serine hydroxymethyltransferase (SHMT) is a key enzyme in the formation and regulation of the folate one-carbon pool. Recent studies on human subjects have shown the existence of two single nucleotide polymorphisms that may be associated with several disease states. One of these mutations results in Ser394 being converted to an Asn (S394N) and the other in the change of Leu474 to a Phe (L474F). These mutations were introduced into the cDNA for both human and rabbit cytosolic SHMT and the mutant enzymes expressed and purified from an Escherichia coli expression system. The mutant enzymes show normal values for kcat and Km for serine. However, the S394N mutant enzyme has increased dissociation constant values for both glycine and tetrahydrofolate (tetrahydropteroylglutamate) and its pentaglutamate form compared to wild-type enzyme. The L474F mutant shows lowered affinity (increased dissociation constant) for only the pentaglutamate form of the folate ligand. Both mutations result in decreased rates of pyridoxal phosphate addition to the mutant apo enzymes to form the active holo enzymes. Neither mutation significantly affects the stability of SHMT or the rate at which it converts 5,10-methenyl tetrahydropteroyl pentaglutamate to 5-formyl tetrahydropteroyl pentaglutamate. Analysis of the structures of rabbit and human SHMT show how mutations at these two sites can result in the observed functional differences.     

A three-dimensional structure of Plasmodium falciparum serine hydroxymethyltransferase in complex with glycine and 5-formyl-tetrahydrofolate. Homology modeling and molecular dynamics

Cytosolic Plasmodium falciparum serine hydroxymethyltransferase (pfSHMT) is a potential target for antimalarial chemotherapy. Contrasting with the other enzymes involved in the parasite folate cycle, little information is available about this enzyme, and its crystallographic structure is unknown yet. In this paper, we propose a theoretical low-resolution 3D model for pfSHMT in complex with glycine and 5-formyl tetrahydrofolate (5-FTHF) based on homology modeling by multiple alignment followed by intensive optimization, validation and dynamics simulations in water. Comparison between the active sites of our model and that of crystallographic Human SHMT (hSHMT) revealed key differences that could be useful for the design of new selective inhibitors of pfSHMT.     

Crystal structure at 2.4 A resolution of E. coli serine hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate

Serine hydroxymethyltransferase (EC 2.1.2.1), a member of the alpha-class of pyridoxal phosphate enzymes, catalyzes the reversible interconversion of serine and glycine, changing the chemical bonding at the C(alpha)-C(beta) bond of the serine side-chain mediated by the pyridoxal phosphate cofactor. Scission of the C(alpha)-C(beta) bond of serine substrate produces a glycine product and most likely formaldehyde, which reacts without dissociation with tetrahydropteroylglutamate cofactor. Crystal structures of the human and rabbit cytosolic serine hydroxymethyltransferases (SHMT) confirmed their close similarity in tertiary and dimeric subunit structure to each other and to aspartate aminotransferase, the archetypal alpha-class pyridoxal 5'-phosphate enzyme. We describe here the structure at 2.4 A resolution of Escherichia coli serine hydroxymethyltransferase in ternary complex with glycine and 5-formyl tetrahydropteroylglutamate, refined to an R-factor value of 17.4 % and R(free) value of 19.6 %. This structure reveals the interactions of both cofactors and glycine substrate with the enzyme. Comparison with the E. coli aspartate aminotransferase structure shows the distinctions in sequence and structure which define the folate cofactor binding site in serine hydroxymethyltransferase and the differences in orientation of the amino terminal arm, the evolution of which was necessary for elaboration of the folate binding site. Comparison with the unliganded rabbit cytosolic serine hydroxymethyltransferase structure identifies changes in the conformation of the enzyme, similar to those observed in aspartate aminotransferase, that probably accompany the binding of substrate. The tetrameric quaternary structure of liganded E. coli serine hydroxymethyltransferase also differs in symmetry and relative disposition of the functional tight dimers from that of the unliganded eukaryotic enzymes. SHMT tetramers have surface charge distributions which suggest distinctions in folate binding between eukaryotic and E. coli enzymes. The structure of the E. coli ternary complex provides the basis for a thorough investigation of its mechanism through characterization and structure determination of site mutants.     

Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folate-independent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme. The cleavage of 3-hydroxy amino acids to glycine and an aldehyde occurs by a retroaldol mechanism. However, the folate-dependent cleavage of serine can be described by either the same retroaldol mechanism with formaldehyde as an enzyme-bound intermediate or by a nucleophilic displacement mechanism in which N5 of tetrahydrofolate displaces the C3 hydroxyl of serine, forming a covalent intermediate. Glu75 of SHMT is clearly involved in the reaction mechanism; it is within hydrogen bonding distance of the hydroxyl group of serine and the formyl group of 5-formyltetrahydrofolate in complexes of these species with SHMT. This residue was changed to Leu and Gln, and the structures, kinetics, and spectral properties of the site mutants were determined. Neither mutation significantly changed the structure of SHMT, the spectral properties of its complexes, or the kinetics of the retroaldol cleavage of allothreonine and 3-phenylserine. However, both mutations blocked the folate-dependent serine-to-glycine reaction and the conversion of methenyltetrahydrofolate to 5-formyltetrahydrofolate. These results clearly indicate that interaction of Glu75 with folate is required for folate-dependent reactions catalyzed by SHMT. Moreover, we can now propose a promising modification to the retroaldol mechanism for serine cleavage. As the first step, N5 of tetrahydrofolate makes a nucleophilic attack on C3 of serine, breaking the C2-C3 bond to form N5-hydroxymethylenetetrahydrofolate and an enzyme-bound glycine anion. The transient formation of formaldehyde as an intermediate is possible, but not required. This mechanism explains the greatly enhanced rate of serine cleavage in the presence of folate, and avoids some serious difficulties presented by the nucleophilic displacement mechanism involving breakage of the C3-OH bond.     

Inhibition of 5,10-methenyltetrahydrofolate synthetase

The interaction of 5-formyltetrahydrofolate analogs with murine methenyltetrahydrofolate synthetase (MTHFS) was investigated using steady-state kinetics, molecular modeling, and site-directed mutagenesis. MTHFS catalyzes the irreversible cyclization of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Folate analogs that cannot undergo the rate-limiting step in catalysis were inhibitors of murine MTHFS. 5-Formyltetrahydrohomofolate was an effective inhibitor of murine MTHFS (K(i)=0.7 microM), whereas 5-formyl,10-methyltetrahydrofolate was a weak inhibitor (K(i)=10 microM). The former, but not the latter, was slowly phosphorylated by MTHFS. 5-Formyltetrahydrohomofolate was not a substrate for murine MTHFS, but was metabolized when the MTHFS active site Y151 was mutated to Ala. MTHFS active site residues do not directly facilitate N10 attack on the on the N5-iminium phosphate intermediate, but rather restrict N10 motion around N5. Inhibitors specifically designed to block N10 attack appear to be less effective than the natural 10-formyltetrahydrofolate polyglutamate inhibitors.     

Crystal structure of rabbit cytosolic serine hydroxymethyltransferase at 2.8 A resolution: mechanistic implications.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to form glycine and single carbon groups that are essential for many biosynthetic pathways. SHMT requires both pyridoxal phosphate (PLP) and tetrahydropteroylpolyglutamate (H4PteGlun) as cofactors, the latter as a carrier of the single carbon group. We describe here the crystal structure at 2.8 A resolution of rabbit cytosolic SHMT (rcSHMT) in two forms: one with the PLP covalently bound as an aldimine to the Nepsilon-amino group of the active site lysine and the other with the aldimine reduced to a secondary amine. The rcSHMT structure closely resembles the structure of human SHMT, confirming its similarity to the alpha-class of PLP enzymes. The structures reported here further permit identification of changes in the PLP group that accompany formation of the geminal diamine complex, the first intermediate in the reaction pathway. On the basis of the current mechanism derived from solution studies and the properties of site mutants, we are able to model the binding of both the serine substrate and the H4PteGlun cofactor. This model explains the properties of several site mutants of SHMT and offers testable hypotheses for a more detailed mechanism of this enzyme.     

Location of the pteroylpolyglutamate-binding site on rabbit cytosolic serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT; EC 2.1.2.1) catalyzes the reversible interconversion of serine and glycine with transfer of the serine side chain one-carbon group to tetrahydropteroylglutamate (H(4)PteGlu), and also the conversion of 5,10-methenyl-H(4)PteGlu to 5-formyl-H(4)PteGlu. In the cell, H(4)PteGlu carries a poly-gamma-glutamyl tail of at least 3 glutamyl residues that is required for physiological activity. This study combines solution binding and mutagenesis studies with crystallographic structure determination to identify the extended binding site for tetrahydropteroylpolyglutamate on rabbit cytosolic SHMT. Equilibrium binding and kinetic measurements of H(4)PteGlu(3) and H(4)PteGlu(5) with wild-type and Lys --> Gln or Glu site mutant homotetrameric rabbit cytosolic SHMTs identified lysine residues that contribute to the binding of the polyglutamate tail. The crystal structure of the enzyme in complex with 5-formyl-H(4)PteGlu(3) confirms the solution data and indicates that the conformation of the pteridine ring and its interactions with the enzyme differ slightly from those observed in complexes of the monoglutamate cofactor. The polyglutamate chain, which does not contribute to catalysis, exists in multiple conformations in each of the two occupied binding sites and appears to be bound by the electrostatic field created by the cationic residues, with only limited interactions with specific individual residues.     

Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes a hydroxymethyl group transfer from l-serine to tetrahydrofolate (H₄folate) to yield glycine and 5,10-methylenetetrahydrofolate (CH₂-H₄folate). SHMT is crucial for deoxythymidylate biosynthesis and a target for antimalarial drug development. Our previous studies indicate that PvSHMT catalyzes the reaction via a ternary complex mechanism. To define the kinetic mechanism of this catalysis, we explored the PvSHMT reaction by employing various methodologies including ligand binding, transient, and steady-state kinetics as well as product analysis by rapid-quench and HPLC/MS techniques. The results indicate that PvSHMT can bind first to either l-serine or H₄folate. The dissociation constants for the enzyme·l-serine and enzyme·H₄folate complexes were determined as 0.18 ± 0.08 and 0.35 ± 0.06 mm, respectively. The amounts of glycine formed after single turnovers of different preformed binary complexes were similar, indicating that the reaction proceeds via a random-order binding mechanism. In addition, the rate constant of glycine formation measured by rapid-quench and HPLC/MS analysis is similar to the k_cat value (1.09 ± 0.05 s⁻¹) obtained from the steady-state kinetics, indicating that glycine formation is the rate-limiting step of SHMT catalysis. This information will serve as a basis for future investigation on species-specific inhibition of SHMT for antimalarial drug development.     

Identification and biochemical characterization of serine hydroxymethyl transferase in the hydrogenosome of Trichomonas vaginalis

Serine hydroxymethyl transferase (SHMT) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. We have identified a single gene encoding SHMT in the genome of Trichomonas vaginalis, an amitochondriate, deep-branching unicellular protist. The protein possesses a putative N-terminal hydrogenosomal presequence and was shown to localize to hydrogensomes by immunofluorescence analysis, providing evidence of amino acid metabolism in this unusual organelle. In contrast to the tetrameric SHMT that exists in the mammalian host, we found that the T. vaginalis SHMT is a homodimer, as found in prokaryotes. All examined SHMT contain an 8-amino-acid conserved sequence, VTTTTHKT, containing the active-site lysyl residue (Lys 251 in TvSHMT) that forms an internal aldimine with PLP. We mutated this Lys residue to Arg and Gln and examined structural and catalytic properties of the wild-type and mutant enzymes in comparison to that reported for the mammalian protein. The oligomeric structure of the mutant K251R and K251Q TvSHMT was not affected, in contrast to that observed for comparable mutations in the mammalian enzyme. Likewise, contrary to that observed for mammalian SHMT, the catalytic activity of K251R TvSHMT was unaffected in the presence of PLP. The K251Q TvSHMT, however, was found to be inactive. These studies indicate that the active site of the parasite enzyme is distinct from its prokaryotic and eukaryotic counterparts and identify TvSHMT as a potential drug target.     

5-Formyltetrahydrofolate polyglutamates are slow tight binding inhibitors of serine hydroxymethyltransferase

The interaction of the mono- and triglutamate forms of 5-methyltetrahydrofolate and 5-formyltetrahydrofolate with serine hydroxymethyltransferase were determined by several methods. These methods included: determining dissociation constants by observing the absorbance at 502 nm of a ternary complex of the enzyme, glycine, and the folate compounds; determining inhibition constants from steady-state reactions; and determining the rate of formation and breakdown of the enzyme inhibitor complex by rapid reaction kinetics. Studies of the dissociation and inhibitor constants showed that both 5-methyltetrahydrofolate and 5-formyltetrahydrofolate have essentially the same affinity for the enzyme-glycine binary complex. However, rapid reaction and steady-state kinetic studies showed that the triglutamate form of 5-formyltetrahydrofolate both binds and is released much more slowly from the enzyme-glycine binary complex, compared with the triglutamate form of 5-methyltetrahydrofolate. The results also showed that only one rotamer of 5-formyltetrahydrofolate binds at the active site of serine hydroxymethyltransferase. The results are discussed in terms of the possible role of 5-formyltetrahydrofolate polyglutamates in regulation of one-carbon metabolism.     

D-serine dehydratase from Escherichia coli. DNA sequence and identification of catalytically inactive glycine to aspartic acid variants

We have identified two glycyl residues whose integrity is essential for the catalytic competence of a model pyridoxal 5'-phosphate requiring enzyme, D-serine dehydratase from Escherichia coli. This was accomplished by isolating and sequencing the structural gene from wild type E. coli and from two mutant strains that produce inactive D-serine dehydratase. DNA sequencing indicated the presence of a single glycine to aspartic acid replacement in each variant. The amino acid replacements lie in a glycine-rich region of D-serine dehydratase well removed from pyridoxal 5'-phosphate-binding lysine 118 in the primary structure of the enzyme. The striking effect of these two glycine to aspartic acid replacements on catalytic activity, the conservation of the glycine-rich region in several pyridoxal 5'-phosphate-dependent enzymes that catalyze alpha/beta-eliminations, and the placement of similar glycine-rich sequences in well-characterized active site structures suggest that the glycine-rich region interacts with the cofactor at the active site of the enzyme.     

Pyridoxal 5'-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase

Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B₆ requiring enzymes. PLP reacts with apo-B₆ enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B₆ enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4′-aldehyde moiety forms covalent adducts with other compounds and non-B₆ proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B₆ enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.     

Regulation of de novo purine biosynthesis by methenyltetrahydrofolate synthetase in neuroblastoma

5-Formyltetrahydrofolate (5-formylTHF) is the only folate derivative that does not serve as a cofactor in folate-dependent one-carbon metabolism. Two metabolic roles have been ascribed to this folate derivative. It has been proposed to 1) serve as a storage form of folate because it is chemically stable and accumulates in seeds and spores and 2) regulate folate-dependent one-carbon metabolism by inhibiting folate-dependent enzymes, specifically targeting folate-dependent de novo purine biosynthesis. Methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme that metabolizes 5-formylTHF and catalyzes its ATP-dependent conversion to 5,10-methenylTHF. This reaction determines intracellular 5-formylTHF concentrations and converts 5-formylTHF into an enzyme cofactor. The regulation and metabolic role of MTHFS in one-carbon metabolism was investigated in vitro and in human neuroblastoma cells. Steady-state kinetic studies revealed that 10-formylTHF, which exists in chemical equilibrium with 5,10-methenylTHF, acts as a tight binding inhibitor of mouse MTHFS. [6R]-10-formylTHF inhibited MTHFS with a K(i) of 150 nM, and [6R,S]-10-formylTHF triglutamate inhibited MTHFS with a K(i) of 30 nm. MTHFS is the first identified 10-formylTHF tight-binding protein. Isotope tracer studies in neuroblastoma demonstrate that MTHFS enhances de novo purine biosynthesis, indicating that MTHFS-bound 10-formylTHF facilitates de novo purine biosynthesis. Feedback metabolic regulation of MTHFS by 10-formylTHF indicates that 5-formylTHF can only accumulate in the presence of 10-formylTHF, providing the first evidence that 5-formylTHF is a storage form of excess formylated folates in mammalian cells. The sequestration of 10-formylTHF by MTHFS may explain why de novo purine biosynthesis is protected from common disruptions in the folate-dependent one-carbon network.     

Comparative aspects of aminooxyacetate inhibition of glycine oxidation and aminotransferase activity of pea leaf mitochondria

Aminooxyacetate (AOA) was found to inhibit glycine oxidation by pea leaf mitochondria at micromolar levels. The inhibition resulted from an inhibition of both glycine decarboxylase and serine hydroxymethyltransferase (SHMT) activity. Aspartate: 2-oxoglutarate aminotransferase (AsAT) and alanine: 2-oxoglutarate aminotransferase activities of pea leaf mitochondria were also very sensitive to AOA inhibition. Inhibition of both glycine oxidation and aminotransferase activity was likely competitive with respect to the amino group substrate, but also displayed a time-dependent increase in inhibition at constant AOA concentration. In the case of glycine oxidation, this time-dependent component may be related to the rate of penetration of AOA across the inner mitochondrial membrane. Furthermore, the AOA-inhibition of glycine oxidation could be reversed by pyridoxal 5-phosphate (PLP), whereas AOA-inhibited aminotransferase activity was not reversed. The results indicate that the pyridoxal 5-phosphate antagonist, AOA, results in varying types of inhibition depending on the type of enzyme involved.     

Formyltetrahydrofolate dehydrogenase-hydrolase from pig liver: simultaneous assay of the activities

The bifunctional folate-dependent enzyme, 10-formyltetrahydrofolate dehydrogenase-hydrolase (10-formyltetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.6), has been purified to homogeneity from pig liver. Its amino acid composition was determined and gave a calculated v of 0.735 ml/g; a molecular weight of 92500 for the protein subunit was determined as well. Spectrophotometric, fluorescence emission and radiochemical methods were devised to assay the activities. Quantitative separation of carbon dioxide and formate produced by the dehydrogenase and the hydrolase reactions, respectively, demonstrated that both activities occur simultaneously. This fact, together with a 5-fold difference in the Km values for the folate substrate, strongly suggests that these two activities are functions of different sites. The possible role of polyglutamate specificity for the preferential selection of one of the activities under physiological conditions was ruled out when both proved to have similar specificities, as determined by sensitivity to inhibition by tetrahydropteroylpolyglutamates.