Mammalian folylpoly-gamma-glutamate synthetase. 2. Substrate specificity and kinetic properties

The specificity of hog liver folylpolyglutamate synthetase for folate substrates and for nucleotide and glutamate substrates and analogues has been investigated. The kinetic mechanism, determined by using aminopterin as the folate substrate, is ordered Ter-Ter with MgATP binding first, folate second, and glutamate last. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. Folate, dihydrofolate, and tetrahydrofolate possess the optimal configurations for catalysis (kcat = 2.5 s-1) while 5- and 10-position substitutions of the folate molecule impair catalysis. kcat values decrease with increasing glutamate chain length, and the rate of decrease varies depending on the state of reduction and substitution of the folate molecule. Folate binding, as assessed by on rates, is slow. Dihydrofolate exhibits the fastest rate, and the rates are slightly reduced for tetrahydrofolate and 10-formyltetrahydrofolate and greatly reduced for 5-methyltetrahydrofolate and folic acid. The on rates for most pteroyldiglutamates are similar to the rates for their respective monoglutamate derivatives, but further extension of the glutamate chain results in a progressive decrease in on rates. Tetrahydrofolate polyglutamates are the only long glutamate chain length folates with detectable substrate activity. The specificity of the L-glutamate binding site is very narrow. L-Homocysteate and 4-threo-fluoroglutamate are alternate substrates and act as chain termination inhibitors in that their addition to the folate molecule prevents or severely retards the further addition of glutamate moieties. The Km for glutamate is dependent on the folate substrate used. MgATP is the preferred nucleotide substrate, and beta,gamma-methylene-ATP, beta,gamma-imido-ATP, adenosine 5'-O-(3-thiotriphosphate), P1,P5-di(adenosine-5') pentaphosphate, and free ATP4- are potent inhibitors of the reaction.     

Mammalian folylpoly-gamma-glutamate synthetase. 4. In vitro and in vivo metabolism of folates and analogues and regulation of folate homeostasis

The regulation of folate and folate analogue metabolism was studied in vitro by using purified hog liver folylpolyglutamate synthetase as a model system and in vivo in cultured mammalian cells. The types of folylpolyglutamates that accumulate in vivo in hog liver, and changes in cellular folate levels and folylpolyglutamate distributions caused by physiological and nutritional factors such as changes in growth rates and methionine, folate, and vitamin B12 status, can be mimicked in vitro by using purified enzyme. Folylpolyglutamate distributions can be explained solely in terms of the substrate specificity of folylpolyglutamate synthetase and can be modeled by using kinetic parameters obtained with purified enzyme. Low levels of folylpolyglutamate synthetase activity are normally required for the cellular metabolism of folates to retainable polyglutamate forms, and consequently folate retention and concentration, while higher levels of activity are required for the synthesis of the long chain length derivatives that are found in mammalian tissues. The synthesis of very long chain derivatives, which requires tetrahydrofolate polyglutamates as substrates, is a very slow process in vivo. The slow metabolism of 5-methyltetrahydrofolate to retainable polyglutamate forms causes the decreased tissue retention of folate in B12 deficiency. Although cellular folylpolyglutamate distributions change in response to nutritional and physiological modulations, it is unlikely that these changes play a regulatory role in one-carbon metabolism as folate distributions respond only slowly. 4-Aminofolates are metabolized to retainable forms at a slow rate compared to folates. Although folate accumulation by cells is not very responsive to changes in folylpolyglutamate synthetase levels and cellular glutamate concentrations, cellular accumulation of anti-folate agents would be highly responsive to any factor that changes the expression of folylpolyglutamate synthetase activity.     

Folylpoly-γ-glutamate synthesis by bacteria and mammalian cells

Summary The purification and properties of folylpolyglutamate synthetase fromCorynebacterium sp, and some properties of partially purified enzyme fromLactobacillus casei, Streptococcus faecalis, Neurospora crassa, pig liver, and Chinese hamster ovary cells, are described.TheCorynebacterium enzyme catalyzes a MgATP-dependent addition of glutamate to a variety of reduced pteroate and pteroylmono-, di-, and triglutamate substrates, with the concomitant production of MgADP and phosphate. Although glutamate moieties are added in a sequential fashion, the kinetic mechanism, which is Ordered Ter Ter, precludes the sequential addition of glutamate moieties to enzyme-bound folate. It is suggested that catalysis precedes via the formation of a pteroyl--glutamyl phosphate intermediate.Thein vivo distribution of folylpolyglutamates in bacteria and mammalian cells, which differ from source to source, appear to be a reflection of the ability of folylpolyglutamates to act as substrates for folylpolyglutamate synthetases from different sources.Only one enzyme appears to be involved in the conversion of pteroylmonoglutamates to polyglutamate forms in both bacteria and mammalian cells. Bacterial folylpolyglutamate synthetases use a variety of pteroylmonoglutamates as their preferred monoglutamate substrate, but use 5,10-methylenetetrahydropteroylpolyglutamates as their preferred, and sometimes only, polyglutamate substrate. Mono- and polyglutamyl forms of tetrahydrofolate are the preferred substrates of mammalian folylpolyglutamate synthetases.     

Mutagenesis of folylpolyglutamate synthetase indicates that dihydropteroate and tetrahydrofolate bind to the same site

The folylpolyglutamate synthetase (FPGS) enzyme of Escherichia coli differs from that of Lactobacillus casei in having dihydrofolate synthetase activity, which catalyzes the production of dihydrofolate from dihydropteroate. The present study undertook mutagenesis to identify structural elements that are directly responsible for the functional differences between the two enzymes. The amino terminal domain (residues 1-287) of the E. coli FPGS was found to bind tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. The domain-swap chimera proteins between the E. coli and the L. casei enzymes possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Recent structural studies have identified two unique folate binding sites, the omega loop in L. casei FPGS and the dihydropteroate binding loop in the E. coli enzyme. Mutants with swapped omega loops retained the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating L. casei FPGS to contain an E. coli FPGS dihydropteroate binding loop did not alter its substrate specificity to using dihydropteroate as a substrate. The mutant D154A, a residue specific for the dihydropteroate binding site in E. coli FPGS, and D151A, the corresponding mutant in the L. casei enzyme, were both defective in using tetrahydrofolate as their substrate, suggesting that the binding site corresponding to the E. coli pteroate binding site is also the tetrahydrofolate binding site for both enzymes. Tetrahydrofolate diglutamate was a slightly less effective substrate than the monoglutamate with the wild-type enzyme but was a 40-fold more effective substrate with the D151A mutant. This suggests that the 5,10-methylenetetrahydrofolate binding site identified in the L. casei ternary structure may bind diglutamate and polyglutamate folate derivatives.     

In Vitro Synthesis of Pteroylpoly-{gamma}-glutamates by Cotyledon Extracts of Pisum sativum L.

The formation of folylpolyglutamate derivatives by germinatingpea seeds (Pisum sativum L. cv Homesteader) was examined invivo and in vitro. Differential microbiological assay of cotyledonextracts showed that total folate concentrations increased from163 ng folate equivalents per g fresh weight at day 1 to 680ng per g fresh weight at day 3 of germination. Over a 7 daygermination period, folylpolyglutamate derivatives accountedfor 46–73% of the total cotyledonary folate pool. Theconcentration of these polyglutamate forms of folate increased6.5 fold during the first four days of germination and thenremained relatively constant. Dialyzed extracts of 1–4 day old cotyledons had abilityto incorporate [³H]glutamate and [¹⁴C]tetrahydrofolate intofolylpolyglutamates. This activity was mainly associated withprotein precipitating at 35–45% of saturation with ammoniumsulphate. The folylpolyglutamate synthetase of pea cotyledonshad requirements for ATP and the monoglutamate of tetrahydrofolate.The latter folate was a more effective substrate than 5,10-methylenetetrahydrofolatebut the diglutamate of unsubstituted tetrahydrofolate was notutilized. Ion exchange chromatography of the reaction productssuggested that [³H]glutamate and [¹⁴C]tetrahydrofolate wereincorporated into di-, and tetraglutamates of tetrahydrofolate.Folates of longer glutamyl chain lengths were only detectedwhen the synthetase reaction proceeded for 12 h or longer. (Received August 23, 1985; Accepted January 22, 1986)     

Concentration-dependent processivity of multiple glutamate ligations catalyzed by folylpoly-gamma-glutamate synthetase

Folylpoly-gamma-glutamate synthetase (FPGS, EC 6.3.2.17) is an ATP-dependent ligase that catalyzes formation of poly-gamma-glutamate derivatives of reduced folates and antifolates such as methotrexate and 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDAH 4PteGlu 1). While the chemical mechanism of the reaction catalyzed by FPGS is known, it is unknown whether single or multiple glutamate residues are added following each folate binding event. A very sensitive high-performance liquid chromatography method has been used to analyze the multiple ligation reactions onto radiolabeled DDAH 4PteGlu 1 catalyzed by FPGS to distinguish between distributive or processive mechanisms of catalysis. Reaction time courses, substrate trapping, and pulse-chase experiments were used to assess folate release during multiple glutamate additions. Together, the results of these experiments indicate that hFPGS can catalyze the processive addition of approximately four glutamate residues to DDAH 4PteGlu 1. The degree of processivity was determined to be dependent on the concentration of the folate substrate, thus suggesting a mechanism for the regulation of folate polyglutamate synthesis in cells.     

Polyglutamylation of folate coenzymes is necessary for methionine biosynthesis and maintenance of intact mitochondrial genome in Saccharomyces cerevisiae

One-carbon metabolism is essential to provide activated one-carbon units in the biosynthesis of methionine, purines, and thymidylate. The major forms of folates in vivo are polyglutamylated derivatives. In organisms that synthesize folate coenzymes de novo, the addition of the glutamyl side chains is achieved by the action of two enzymes, dihydrofolate synthetase and folylpolyglutamate synthetase. We report here the characterization and molecular analysis of the two glutamate-adding enzymes of Saccharomyces cerevisiae. We show that dihydrofolate synthetase catalyzing the binding of the first glutamyl side chain to dihydropteroate yielding dihydrofolate is encoded by the YMR113w gene that we propose to rename FOL3. Mutant cells bearing a fol3 mutation require folinic acid for growth and have no dihydrofolate synthetase activity. We show also that folylpolyglutamate synthetase, which catalyzes the extension of the glutamate chains of the folate coenzymes, is encoded by the MET7 gene. Folylpolyglutamate synthetase activity is required for methionine synthesis and for maintenance of mitochondrial DNA. We have tested whether two folylpolyglutamate synthetases could be encoded by the MET7 gene, by the use of alternative initiation codons. Our results show that the loss of mitochondrial functions in met7 mutant cells is not because of the absence of a mitochondrial folylpolyglutamate synthetase.     

Substrate specificity of mammalian folylpoly-gamma-glutamate synthetase for 5,8-dideazafolates and 5,8-dideaza analogues of aminopterin

The substrate specificity of pig liver folylpolyglutamate synthetase (tetrahydrofolate:L-glutamate gamma-ligase (ADP-forming), EC 6.3.2.17) for classical 5,8-dideaza analogues of folic acid, isofolic acid aminopterin and isoaminopterin has been investigated. 5,8-Dideazafolate and 5,8-dideazaaminopterin are very effective substrates with activities approaching those of the best reduced folate substrates. The analogous isofolate analogues are less effective substrates, but still better than folic acid. The 5-chloro substituent is the only modification that consistently increases the on rate, with 5-chloro-5,8-dideazaaminopterin being the most effective substrate found, thus far, for the enzyme. Methylation at positions 9 or 10 generally decreases binding, while 5-methylation increases the binding of 4-oxoquinazolines, but decreases the binding of their 4-amino counterparts. The presence of a formyl group at N9 or N10 has the opposite effect, decreasing the binding of 4-oxo analogues while increasing the rate for 4-amino derivatives. Increases in on rate with methyl, formyl or 4-amino substitutions are only significant when the parent compound is a poor substrate, suggesting that these groups do not interact directly with the enzyme but cause conformational changes in the structure of the substrate that influence binding to the enzyme.     

Enzymatic synthesis and function of folylpolyglutamates

Summary Derivatives of folic acid occur in nature predominantly as poly (-glutamyl) derivatives containing 2–8 glutamate residues. The data regarding the function of these derivatives, and their biosynthesis by eucaryotic and procaryotic folylpolyglutamate synthetases, is reviewed.The most universal functions of folylpolyglutamates appear to be (a) as the actual cofactors in vivo for folate dependent enzymes, (b) as inhibitors of folate dependent enzymes for which they are not substrates, and (c) to increase retention of folates after they are transported into cells as monoglutamates. Folylpolyglutamates also have numerous specialized functions in specific organisms, e.g. as structural components of some coliphage, and as allosteric regulators in Neurospora crassa.A single enzyme appears responsible for synthesis of all polyglutamate derivatives, regardless of length. With the recent introduction of sensitive assays this folylpolyglutamate synthetase has begun to be characterized. Although procaryotic and eucaryotic synthetases have many dissimilar properties, both types catalyze the ATP-dependent addition of L-glutamate to the -carboxyl of the glutamate present in the folate. Both types also require a monovalent cation and a relatively high pH. The most significant differences between the two types are in their folate substrate specificity and the product lengths derived from various folates.The mechanism of the bacterial enzyme has been studied and an acyl phosphate intermediate is indicated.     

In vitro synthesis of alpha-carboxyl-linked folylpolyglutamates by an enzyme preparation from Escherichia coli

Extracts of Escherichia coli contained an enzymatic activity which catalyzed the addition of L-glutamate to the alpha-carboxyl of various polyglutamate substrates, including folylpolyglutamates. Much of the enzyme activity was separated by DE52 chromatography and gel filtration from the enzyme which adds the first three glutamates in the biosynthesis of folylpolyglutamates, dihydrofolate synthetase-folylpolyglutamate synthetase. The two enzyme activities differed in many properties. Whereas dihydrofolate synthetase-folylpolyglutamate synthetase preferred pteroate or pteroylmonoglutamate substrates, the folylpoly-alpha-glutamate synthetase preparations effectively utilized tetrahydropteroylpolyglutamates, pteroylpolyglutamates, p-aminobenzoylpolyglutamates (pAB(Glu)n), and even a polyglutamate tripeptide. Several di- and triglutamyl peptides were inhibitory to folylpoly-alpha-glutamate synthetase activity, but not to dihydrofolate synthetase-folylpolyglutamate synthetase. Conversely, dihydropteroate noncompetitively inhibits the folylpolyglutamate synthetase reaction of the dihydrofolate synthetase-folylpolyglutamate synthetase protein, but did not inhibit the folylpoly-alpha-glutamate synthetase reaction. Potassium chloride was inhibitory to folylpoly-alpha-glutamate synthetase activity (as were other salts and several polyanions), in contrast to the absolute requirement of dihydrofolate synthetase-folylpolyglutamate synthetase activity for a monovalent cation such as K+. Incubation of a folylpoly-alpha-glutamate synthetase preparation with (6S)-tetrahydropteroyltri(gamma)glutamate generated products which after chemical cleavage to pAB(Glu)n were identical to those from growing E. coli, in high performance liquid chromatography retention times and in pattern of digestion by alpha-COOH bond-specific carboxypeptidase Y. High performance liquid chromatography and mass spectral analysis of the products of the in vitro reactions of folylpoly-alpha-glutamate synthetase with several substrates also demonstrated the addition of glutamate residues via alpha-COOH linkages. Thus, there appear to be two folylpolyglutamate synthetase activities in E. coli, dihydrofolate synthetase-folylpolyglutamate synthetase which adds the first three glutamate residues by gamma-COOH linkages and the folylpoly-alpha-glutamate synthetase activity which extends the folylpolyglutamate chain via gamma-COOH peptide bonds.     

Folate-binding triggers the activation of folylpolyglutamate synthetase

Folic acid is an essential vitamin for normal cell growth, primarily through its central role in one-carbon metabolism. Folate analogs (antifolates) are targeted at the same reactions and are widely used as therapeutic drugs for cancer and bacterial infections. Effective retention of folates in cells and the efficacy of antifolate drugs both depend upon the addition of a polyglutamate tail to the folate or antifolate molecule by the enzyme folylpolyglutamate synthetase (FPGS). The reaction mechanism involves the ATP-dependent activation of the free carboxylate group on the folate molecule to give an acyl phosphate intermediate, followed by attack by the incoming L-glutamate substrate. FPGS shares a number of structural and mechanistic details with the bacterial cell wall ligases MurD, MurE and MurF, and these enzymes, along with FPGS, form a subfamily of the ADP-forming amide bond ligase family. High-resolution crystallographic analyses of binary and ternary complexes of Lactobacillus casei FPGS reveal that binding of the first substrate (ATP) is not sufficient to generate an active enzyme. However, binding of folate as the second substrate triggers a large conformational change that activates FPGS and allows the enzyme to adopt a form that is then able to bind the third substrate, L-glutamate, and effect the addition of a polyglutamate tail to the folate.     

Structural basis for recognition of polyglutamyl folates by thymidylate synthase.

Thymidylate synthase (TS) catalyzes the final step in the de novo synthesis of thymidine. In vivo TS binds a polyglutamyl cofactor, polyglutamyl methylenetetrahydrofolate (CH2-H4folate), which serves as a carbon donor. Glutamate residues on the cofactor contribute as much as 3.7 kcal to the interaction between the cofactor, substrate, and enzyme. Because many ligand/receptor interactions appear to be driven largely by hydrophobic forces, it is surprising that the addition of hydrophilic, soluble groups such as glutamates increases the affinity of the cofactor for TS. The structure of a polyglutamyl cofactor analog bound in ternary complex with deoxyuridine monophosphate (dUMP) and Escherichia coli TS reveals how the polyglutamyl moiety is positioned in TS and accounts in a qualitative way for the binding contributions of the different individual glutamate residues. The polyglutamyl moiety is not rigidly fixed by its interaction with the protein except for the first glutamate residue nearest the p-aminobenzoic acid ring of folate. Each additional glutamate is progressively more disordered than the previous one in the chain. The position of the second and third glutamate residues on the protein surface suggests that the polyglutamyl binding site could be utilized by a new family of inhibitors that might fill the binding area more effectively than polyglutamate.     

Studies on the regulatory properties of the pterin cofactor and dopamine bound at the active site of human phenylalanine hydroxylase.

The catalytic activity of phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase EC 1.14.16.1) is regulated by three main mechanisms, i.e. substrate (l-phenylalanine, L-Phe) activation, pterin cofactor inhibition and phosphorylation of a single serine (Ser16) residue. To address the molecular basis for the inhibition by the natural cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, its effects on the recombinant tetrameric human enzyme (wt-hPAH) was studied using three different conformational probes, i.e. the limited proteolysis by trypsin, the reversible global conformational transition (hysteresis) triggered by L-Phe binding, as measured in real time by surface plasmon resonance analysis, and the rate of phosphorylation of Ser16 by cAMP-dependent protein kinase. Comparison of the inhibitory properties of the natural cofactor with the available three-dimensional crystal structure information on the ligand-free, the binary and the ternary complexes, have provided important clues concerning the molecular mechanism for the negative modulatory effects. In the binary complex, the binding of the cofactor at the active site results in the formation of stabilizing hydrogen bonds between the dihydroxypropyl side-chain and the carbonyl oxygen of Ser23 in the autoregulatory sequence. L-Phe binding triggers local as well as global conformational changes of the protomer resulting in a displacement of the cofactor bound at the active site by 2.6 A (mean distance) in the direction of the iron and Glu286 which causes a loss of the stabilizing hydrogen bonds present in the binary complex and thereby a complete reversal of the pterin cofactor as a negative effector. The negative modulatory properties of the inhibitor dopamine, bound by bidentate coordination to the active site iron, is explained by a similar molecular mechanism including its reversal by substrate binding. Although the pterin cofactor and the substrate bind at distinctly different sites, the local conformational changes imposed by their binding at the active site have a mutual effect on their respective binding affinities.     

Reconstitution of Escherichia coli DNA photolyase with various folate derivatives

DNA photolyase from Escherichia coli contains both flavin and pterin. However, the isolated enzyme is depleted with respect to the pterin chromophore (0.5 mol of pterin/mol of flavin). The extinction coefficient of the pterin chromophore at 360 nm is underestimated by a method used in earlier studies which assumes stoichiometric amounts of pterin and flavin. The extinction coefficient of the pterin chromophore, determined on the basis of its (p-aminobenzoyl)polyglutamate content (epsilon 360 = 25.7 x 10(3) M-1 cm-1), is in good agreement with that expected for a 5,10-methenyltetrahydrofolate derivative. Also consistent with this structure, the pterin chromophore could be reversibly hydrolyzed to yield a 10-formyltetrahydrofolate derivative or reduced to yield a 5-methyltetrahydrofolate derivative. The isolated enzyme could be reconstituted with various folate derivatives to yield enzyme that contained equimolar amounts of pterin and flavin. Similar results were obtained in reconstitution studies with the natural pterin chromophore, with 5,10-methenyltetrahydrofolate, and with 10-formyltetrahydrofolate. The results show that the polyglutamate moiety, previously identified in the natural chromophore, is not critical for binding. Reconstitution with the natural pterin chromophore did not affect catalytic activity. The latter is consistent with our previous studies which show that, although the pterin chromophore acts as a sensitizer in native enzyme, it is not essential for dimer repair which can occur at the same rate under saturating light with flavin (1,5-dihydro-FAD) as the only chromophore.     

Folate polyglutamate synthetase activity in the cobalamin-inactivated rat.

Exposure to N2O inactivates cob[I]alamin and interferes with the activity of methionine synthetase, of which cob[I]alamin is a coenzyme. Less directly, it stops the formation of folate polyglutamate from tetrahydrofolates. Studies on the activity of folate polyglutamate synthetase in rat liver in vivo were carried out. The synthetase activity increased after exposure to N2O for up to 48 h, but longer exposure was accompanied by a return of activity to baseline values. The rise in synthetase activity was prevented by supplying methionine, 5'-methylthioadenosine or 5-formyltetrahydrofolate. The fall in folate polyglutamate synthetase activity after 48 h was accompanied by a restoration of hepatic synthesis of folate polyglutamate despite continuation of N2O exposure.     

An inverse relationship of rat liver folate polyglutamate chain length to nutritional folate sufficiency

The relative concentrations of folylpolyglutamates of differing chain length in rat liver and the uptake of exogenous [3H]folic acid (20 microCi, 20 microgram) into liver folylpolyglutamates were examined in rats maintained on (a) standard and folate-supplemented standard diets and (b) semi-defined folate-sufficient and folate-deficient diets. Folylpolyglutamates extracted from liver were cleaved to p-aminobenzoylpolyglutamates which were separated by ion-exchange chromatography. The relative concentrations and ultimate radiolabeling of longer-chain folylpolyglutamates (six, seven and eight glutamate residues) were greatest in the livers of folate-deficient rats, whereas the intermediate-chain folylpolyglutamates (three, four and five glutamate residues) were the greatest portion of total liver folates of folate-supplemented rats. Thus, the length of the polyglutamate chain added to liver folates is inversely related to the total concentration of liver folates. These data suggest that folylpolyglutamate biosynthesis in the liver may be controlled by the liver folate concentrations. In folate insufficiency such a control mechanism would serve to enhance the affinity of folates for folate-dependent enzymes and to conserve the liver folate concentration.     

Mammalian folylpoly-gamma-glutamate synthetase. 1. Purification and general properties of the hog liver enzyme

Folylpolyglutamate synthetase was purified 30,000-150,000-fold from hog liver. Purification required the use of protease inhibitors, and the protein was purified to homogeneity in two forms. Both forms of the enzyme were monomers of Mr 62,000 and had similar specific activities. The specific activity of the homogeneous protein was over 2000-fold higher than reported for partially purified folylpolyglutamate synthetases from other mammalian sources. Enzyme activity was absolutely dependent on the presence of a reducing agent and a monovalent cation, of which K+ was most effective. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to tetrahydrofolate with the concomitant stoichiometric formation of MgADP and phosphate. Under conditions that resembled the expected substrate and enzyme concentrations in hog liver, tetrahydrofolate was metabolized to long glutamate chain length derivatives with the hexaglutamate, the major in vivo folate derivative, predominating. Enzyme activity was maximal at about pH 9.5. The high-pH optimum was primarily due to an increase in the Km value for the L-glutamate substrate at lower pH values, and the reaction proceeded effectively at physiological pH provided high levels of glutamate were supplied.     

Regulation of folylpoly-gamma-glutamate synthesis in bacteria: in vivo and in vitro synthesis of pteroylpoly-gamma-glutamates by Lactobacillus casei and Streptococcus faecalis

Lactobacillus casei and Streptococcus faecalis accumulated labeled folic acid and metabolized this compound to poly-gamma-glutamates of chain lengths of up to 11 and 5, respectively. Octa- and nonaglutamates predominated in L. casei, and tetraglutamates predominated in S. faecalis. The most effective monoglutamate substrates for the L. casei and S. faecalis folylpoly-gamma-glutamate (folylpolyglutamate) synthetases were methylene- and formyltetrahydrofolate, respectively. Methylenetetrahydropteroylpoly-gamma-glutamates were the preferred poly-gamma-glutamate substrates for both enzymes and, in each case, the highest activity was observed with the diglutamate substrate. The final distribution of folylpolyglutamates in these bacteria appeared to reflect the ability of folates with various glutamate chain lengths to act as substrates for the bacterial folylpolyglutamate synthetases. The proportions of individual folylpolyglutamates were markedly affected by culturing the bacteria in medium containing adenine, whereas thymine was without effect. Adenine did not affect the level of folylpolyglutamate synthetase in either organism but caused a large increase in the proportion of intracellular folates containing one-carbon units at the oxidation level of formate, folates which are substrates for enzymes involved in purine biosynthesis. The folates with shorter glutamate chain lengths in bacteria cultured in the presence of adenine resulted from primary regulation of the de novo purine biosynthetic pathway, regulation which caused an accumulation of formyltetrahydropteroyl-poly-gamma-glutamates (folate derivatives that are ineffective substrates for folylpolyglutamate synthetases), and did not result from regulation of folylpolyglutamate synthetase per se.     

Identification of the folate binding sites on the Escherichia coli T-protein of the glycine cleavage system.

T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. To determine the folate-binding site on the enzyme, 14C-labeled methylenetetrahydropteroyltetraglutamate (5,10-CH2-H4PteGlu4) was enzymatically synthesized from methylenetetrahydrofolate (5, 10-CH2-H4folate) and [U-14C]glutamic acid and subjected to cross-linking with the recombinant Escherichia coli T-protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker between amino and carboxyl groups. The cross-linked product was digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase high performance liquid chromatography. Amino acid sequencing of the labeled peptides revealed that three lysine residues at positions 78, 81, and 352 were involved in the cross-linking with polyglutamate moiety of 5, 10-CH2-H4PteGlu4. The comparable experiment with 5,10-CH2-H4folate revealed that Lys-81 and Lys-352 were also involved in cross-linking with the monoglutamate form. Mutants with single or multiple replacement(s) of these lysine residues to glutamic acid were constructed by site-directed mutagenesis and subjected to kinetic analysis. The single mutation of Lys-352 caused similar increase (2-fold) in Km values for both folate substrates, but that of Lys-81 affected greatly the Km value for 5,10-CH2-H4PteGlu4 rather than for 5,10-CH2-H4folate. It is postulated that Lys-352 may serve as the primary binding site to alpha-carboxyl group of the first glutamate residue nearest the p-aminobenzoic acid ring of 5,10-CH2-H4folate and 5,10-CH2-H4PteGlu4, whereas Lys-81 may play a key role to hold the second glutamate residue through binding to alpha-carboxyl group of the second glutamate residue.