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.     

Substrate activity of synthetic formyl phosphate in the reaction catalyzed by formyltetrahydrofolate synthetase

Formyl phosphate, a putative enzyme-bound intermediate in the reaction catalyzed by formyltetrahydrofolate synthetase (EC 6.3.4.3), was synthesized from formyl fluoride and inorganic phosphate [Jaenicke, L. v., & Koch, J. (1963) Justus Liebigs Ann. Chem. 663, 50-58], and the product was characterized by 31P, 1H, and 13C nuclear magnetic resonance (NMR). Measurement of hydrolysis rates by 31P NMR indicates that formyl phosphate is particularly labile, with a half-life of 48 min in a buffered neutral solution at 20 degrees C. At pH 7, hydrolysis occurs with P-O bond cleavage, as demonstrated by 18O incorporation from H2(18)O into Pi, while at pH 1 and pH 13 hydrolysis occurs with C-O bond cleavage. The substrate activity of formyl phosphate was tested in the reaction catalyzed by formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum. Formyl phosphate supports the reaction in both the forward and reverse directions. Thus, N10-formyltetrahydrofolate is produced from tetrahydrofolate and formyl phosphate in a reaction mixture that contains enzyme, Mg(II), and ADP, and ATP is produced from formyl phosphate and ADP with enzyme, Mg(II), and tetrahydrofolate present. The requirements for ADP and for tetrahydrofolate as cofactors in these reactions are consistent with previous steady-state kinetic and isotope exchange studies, which demonstrated that all substrate subsites must be occupied prior to catalysis. The k cat values for both the forward and reverse directions, with formyl phosphate as the substrate, are much lower than those for the normal forward and reverse reactions. Kinetic analysis of the formyl phosphate supported reactions indicates that the low steady-state rates observed for the synthetic intermediate are most likely due to the sequential nature of the normal reaction.     

Formation and utilization of formyl phosphate by N10-formyltetrahydrofolate synthetase: evidence for formyl phosphate as an intermediate in the reaction

N10-Formyltetrahydrofolate synthetase from bacteria and yeast catalyzes a slow formate-dependent ADP formation in the absence of H4folate. The synthesis of formyl phosphate by the enzyme was detected by trapping the intermediate as formyl hydroxamate. That the "formate kinase" activity was part of the catalytic center of N10-formyltetrahydrofolate synthetase was shown by demonstrating coordinate inactivation of the "kinase" and synthetase activities by heat and a sulfhydryl reagent, similar effects of monovalent cations, similar Km values for substrates, and similar Ki values for the inhibitor phosphonoacetaldehyde for both activities. The relative rates of the kinase activities for the bacterial and yeast enzymes are about 10(-4) and 4 x 10(-6) of their respective synthetase activities. These slow rates for the kinase reaction can be explained by the slow dissociation of ADP and formyl phosphate from the enzyme. This conclusion is supported by rapid-quench studies where a "burst" of ADP formation (6.4 s-1) was observed that is considerably faster than the steady-state rate (0.024 s-1). The demonstration of enzyme-bound products by a micropartition assay and the lack of a significant formate-stimulated exchange between ADP and ATP provide further evidence for the slow release of the products from the enzyme. The synthesis of N10-CHO-H4folate when H4folate was added to the E-formyl phosphate-ADP complex is also characterized by a "burst" of product formation. The rate of this burst phase at 5 degrees C occurs with a rate constant of 18 s-1 compared to 14 s-1 for the overall reaction at the same temperature. These results provide further evidence for formyl phosphate as an intermediate in the reaction and are consistent with the sequential mechanism of the normal catalytic pathway. Positional isotope exchange experiments using [beta,gamma-18O]ATP showed no evidence for exchange during turnover experiments in the presence of either H4folate or the competitive inhibitor pteroyltriglutamate. The absence of scrambling of the 18O label as observed by 31P NMR suggests that the central complex may impose restraints to limit free rotation of the P beta oxygens of the product ADP.     

A binding assay for serine hydroxymethyltransferase

A sensitive assay for measuring serine hydroxymethyltransferase activity has been developed, based on the binding of N5,N10-[14C]methylene tetrahydrofolate (THF) to DEAE-cellulose paper. The complete assay requires THF, pyridoxal 5'-phosphate, [14C]serine, and enzyme. The reaction is stopped by streaking an aliquot of the reaction mixture onto a square of DEAE-cellulose paper, washing the paper with water to remove unreacted serine, drying the paper, and counting the bound N5,N10-[14C]methylene-THF. To determine that the labeled product was N5,N10-methylene-THF, unlabeled formaldehyde, which exchanges with the labeled methylene carbon, was added after the product had accumulated; 2 min after the addition of formaldehyde the amount of labeled product was reduced by 50%, and by 85% after 10 min. In addition, glycine, which reverses the reaction, and hydroxylamine, which reacts with the methylene carbon, reduced the number of counts bound to the paper. Binding of product to the filter is proportional to both enzyme concentration and assay time. No counts were retained on phosphocellulose filters. This assay represents a new and simple method for measuring serine hydroxymethyltransferase activity, which can be used to measure enzyme activity in tissue homogenates and for screening large numbers of samples.     

A formyltransferase required for polymyxin resistance in Escherichia coli and the modification of lipid A with 4-Amino-4-deoxy-L-arabinose. Identification and function oF UDP-4-deoxy-4-formamido-L-arabinose

Modification of the phosphate groups of lipid A with 4-amino-4-deoxy-L-arabinose (L-Ara4N) is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium. We previously demonstrated that the enzyme ArnA catalyzes the NAD+-dependent oxidative decarboxylation of UDP-glucuronic acid to yield the UDP-4'-ketopentose, uridine 5'-diphospho-beta-(L-threo-pentapyranosyl-4'-ulose), which is converted by ArnB to UDP-beta-(L-Ara4N). E. coli ArnA is a bi-functional enzyme with a molecular mass of approximately 74 kDa. The oxidative decarboxylation of UDP-glucuronic acid is catalyzed by the 345-residue C-terminal domain of ArnA. The latter shows sequence similarity to enzymes that oxidize the C-4' position of sugar nucleotides, like UDP-galactose epimerase, dTDP-glucose-4,6-dehydratase, and UDP-xylose synthase. We now show that the 304-residue N-terminal domain catalyzes the N-10-formyltetrahydrofolate-dependent formylation of the 4'-amine of UDP-L-Ara4N, generating the novel sugar nucleotide, uridine 5'-diphospho-beta-(4-deoxy-4-formamido-L-arabinose). The N-terminal domain is highly homologous to methionyl-tRNA(f)Met formyltransferase. The structure of the formylated sugar nucleotide generated in vitro by ArnA was validated by 1H and 13C NMR spectroscopy. The two domains of ArnA were expressed independently as active proteins in E. coli. Both were required for maintenance of polymyxin resistance and L-Ara4N modification of lipid A. We conclude that N-formylation of UDP-L-Ara4N is an obligatory step in the biosynthesis of L-Ara4N-modified lipid A in polymyxin-resistant mutants. We further demonstrate that only the formylated sugar nucleotide is converted in vitro to an undecaprenyl phosphate-linked form by the enzyme ArnC. Because the L-Ara4N unit attached to lipid A is not derivatized with a formyl group, we postulate the existence of a deformylase, acting later in the pathway.     

Alkaline opening of imidazole ring of 7-methylguanosine. 2. Further studies on reaction mechanisms and products

High performance liquid chromatography (HPLC) was used to follow the kinetics of the alkaline induced opening of the imidazole ring of 7-methylguanosine (7-meGuo). The kinetics show an initial rapid formation of a major transient intermediate and some minor products that were chromato-graphically separable into seven peaks. This phase of the reaction is followed by the formation of a dominant pyrimidine derivative whose liquid chromatography retention time in a 6% methanol, 0.01 M NH₄H₂PO₄ (pH 5.1) solvent is 6 min; during the rest of the reaction time this dominant species was progressively converted to a co-dominant species that has a 4.5-min column retention. Mass spectroscopy confirmed the existence of two species of ring opened 7-methylguanine (7-meGua), one formylated and another deformylated. Schiff's reaction demonstrated that the species in the second HPLC peak is the formylated one. The ring opened 7-methylguanine (rom⁷Gua) released by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylated species. These results demonstrate that the enzyme excises formylated rom⁷Gua from DNA Analysis of rom⁷Guo by NMR showed that there are two signals assignable to methyl protons and two to formyl protons. These chemical shifts were interpreted as being due to the opening of the imidazole ring at two sites and to the formation of formylated and deformylated rom⁷Gua.     

Structure of a murine cytoplasmic serine hydroxymethyltransferase quinonoid ternary complex: evidence for asymmetric obligate dimers

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single carbon units for purine, thymidine, and methionine biosynthesis. The enzyme is a homotetramer comprising two obligate dimers and four pyridoxal phosphate-bound active sites. The mammalian enzyme is present in cells in both catalytically active and inactive forms. The inactive form is a ternary complex that results from the binding of glycine and 5-formyltetrahydrofolate polyglutamate, a slow tight-binding inhibitor. The crystal structure of a close analogue of the inactive form of murine cytoplasmic SHMT (cSHMT), lacking only the polyglutamate tail of the inhibitor, has been determined to 2.9 A resolution. This first structure of a ligand-bound mammalian SHMT allows identification of amino acid residues involved in substrate binding and catalysis. It also reveals that the two obligate dimers making up a tetramer are not equivalent; one can be described as "tight-binding" and the other as "loose-binding" for folate. Both active sites of the tight-binding dimer are occupied by 5-formyltetrahydrofolate (5-formylTHF), whose N5-formyl carbon is within 4 A of the glycine alpha-carbon of the glycine-pyridoxal phosphate complex; the complex appears to be primarily in its quinonoid form. In the loose-binding dimer, 5-formylTHF is present in only one of the active sites, and its N5-formyl carbon is 5 A from the glycine alpha-carbon. The pyridoxal phosphates appear to be primarily present as geminal diamine complexes, with bonds to both glycine and the active site lysine. This structure suggests that only two of the four catalytic sites on SHMT are catalytically competent and that the cSHMT-glycine-5-formylTHF ternary complex is an intermediate state analogue of the catalytic complex associated with serine and glycine interconversion.     

Rat intestinal phosphodiesterase II. Properties of the highly purified enzyme and its inactivation by iodoacetic acid.

A highly purifed preparation of rat intestinal phosphodiesterase II (oligonucleate 3'-nucleotidohydrolase, EC 3.1.4.18) has been studied using a synthetic substrate, thymidine 3'(2,4-dinitrophenyl) phosphate. The enzyme was most active between pH 6.1 and pH 6.7 and was inhibited by Cu2+ and Zn2+ but unaffected by EDTA, Mg2+, Co2+, and Ni2+. The reaction rate decreased at high levels of enzyme because of competitive inhibition by deoxythymidine 3'-phosphate, a reaction product, which showed a Ki of 2-10(-5) M. The molecular weight of the enzyme by gel-filtration was 150 000-170 000. In electrofocusing experiments multiple peaks of activity were found at pH 3.4, 4.2-4.5and 7.2. Polyacrylamide gel electrophoresis of freshly purified phosphodiesterase II showed up to 10 protein bands in the gels. If the preparations were stored at 4 degrees C for some time only one or two bands appeared. Investigation of the reaction of rat intestinal phosphodiesterase II with a number of possible phosphodiesterase substrates indicated that the enzyme required a nucleoside 3'-phosphoryl residue for the initiation of hydrolysis. Thus compounds such as NAD, ATP, bis-(p-nitrophenyl)phosphate, thymidine 5'-(p-nitrophenyl)phosphate, glycerylphosphorylcholine, guanylyl-(2' leads to 5')-adenosine and 3',5'-cyclic AMP which contain phosphodiester bonds, nevertheless were not substrates for the enzyme. The enzyme was inhibited reverisbly by p-chloromercuribenzoate and p-chloromercuriphenylsulfonate and inactivated irreversibly by iodoacetic acid. Activity of the phosphodiesterase II was reduced to 50% by incubation with 2.0-10(-3)--5.0-10(-3) M iodoacetate for 20--30 min at 24 degrees C at pH 5.0--6.1. Iodoacetamide had no effect. The degree of inactivation by iodoacetate was reduced by the presence of a substrate for the enzyme or, more effectively by deoxythymidine 3'-phosphate, a competitive inhibitor. It is concluded that iodoacetic acid alkylates an essential residue at the active centre of the enzyme.     

Quantification of key folate forms in serum using stable-isotope dilution ultra performance liquid chromatography–tandem mass spectrometry

Folates act as essential coenzymes in many biological pathways. Alteration in folate form distribution might have biological significance, especially in relation to certain genetic polymorphisms. We developed a stable-isotope dilution ultra performance liquid chromatography–mass spectrometry (UPLC–MS/MS) method for quantification of the folate forms 5-methyltetrahydrofolate (5-methylTHF), 5-formylTHF, 5,10-methenylTHF, THF, and folic acid in serum. After extraction using an ion exchange and mixed mode solid-phase, samples were separated and detected using an UPLC–MS/MS system. The quantification limits were between 0.17 nmol/L (5-formylTHF) and 1.79 nmol/L (THF), and the assay was linear up to 100 nmol/L (5-methylTHF) and 10 nmol/L (5-formylTHF, 5,10-methenylTHF, THF, and folic acid). The intraassay CVs for 5-methylTHF and 5-formylTHF were 2.0% and 7.2%, respectively. Mean recoveries were between 82.3% for THF and 110.8% for 5,10-methenylTHF. Concentrations of total folate measured by the new method showed a strong correlation with those measured by an immunologic assay (r = 0.939; p < 0.001). The mean total folate from 32 apparently healthy subjects was 18.09 nmol/L, of which 87.23% was 5-methylTHF. Concentrations of homocysteine showed a better correlation to the total folate measured by the new method compared to that obtained by an immunologic assay. We also confirmed that MTHFR polymorphism has a significant effect on folate distribution in this small population of non-supplemented subjects.     

Change in effective pH of salt solutions on freezing, as evidenced by altered reactivities of heme alpha towards carbonyl reagents.

Addition of NaHSO3 or HCN to the formyl group of heme alpha was greatly accelerated by freezing reaction mixtures prepared in aq. Na2CO3, and freezing resulted in characteristic color and spectral changes of the solutions. Similar changes were observed on decreasing the pH of alkaline reaction mixtures with HCl at room temperature, indicating that the effective pH of certain salt solutions is greatly lowered by freezing. The reactivity of the formyl group changed depending on the redox state of the heme iron and the species of ligand.     

Effect of Asp-235-->Asn substitution on the absorption spectrum and hydrogen peroxide reactivity of cytochrome c peroxidase.

The spectroscopic properties of a mutant cytochrome c peroxidase, in which Asp-235 has been replaced by an asparagine residue, were examined in both nitrate and phosphate buffers between pH 4 and 10.5. The spin state of the enzyme is pH dependent, and four distinct spectroscopic species are observed in each buffer system: a predominantly high-spin Fe(III) species at pH 4, two distinct low-spin forms between pH 5 and 9, and the denatured enzyme above pH 9.3. The spectrum of the mutant enzyme at pH 4 is dependent upon specific ion effects. Increasing the pH above 5 converts the mutant enzyme to a predominantly low-spin hydroxy complex. Subsequent conversion to a second low-spin form is essentially complete at pH 7.5. The second low-spin form has the distal histidine, His-52, coordinated to the heme iron. To evaluate the effect of the changes in coordination state upon the reactivity of the enzyme, the reaction between hydrogen peroxide and the mutant enzyme was also examined as a function of pH. The reaction of CcP(MI,D235N) with peroxide is biphasic. At pH 6, the rapid phase of the reaction can be attributed to the bimolecular reaction between hydrogen peroxide and the hydroxy-ligated form of the mutant enzyme. Despite the hexacoordination of the heme iron in this form, the bimolecular rate constant is approximately 22% that of pentacoordinate wild-type yeast cytochrome c peroxidase. The bimolecular reaction of the mutant enzyme with peroxide exhibits the same pH dependence in nitrate-containing buffers that has been described for the wild-type enzyme, indicating a loss of reactivity with the protonation of a group with an apparent pKa of 5.4. This observation eliminates Asp-235 as the source for this heme-linked ionization and strengthens the hypothesis that the pKa of 5.4 is associated with His-52. The slower phase of the reaction between peroxide and the mutant enzyme saturates at high peroxide concentration and is attributed to conversion of unreactive to reactive forms of the enzyme. The fraction of enzyme which reacts via the slow phase is dependent upon both pH and specific ion effects.     

Purification and characterization of acid phosphatase-1 from Drosophila melanogaster

Acid phosphatase-1 (orthophosphoric monoester phosphohydrolase, acid optimum, EC 3.1.3.2), the major phosphatase in adult Drosophila melanogaster, has been purified to apparent homogeneity. The final product is a glycoprotein homodimer with a subunit molecular weight of about 50,000, as measured by its electrophoretic mobility in denaturing conditions on polyacrylamide gels containing sodium dodecyl sulfate. It has a turnover number of 1720 1-naphthyl phosphate molecules hydrolyzed/s by each acid phosphatase-1 molecule at 37 degrees C, pH 5.0. An average fly contains about 5 ng of enzyme. Pure acid phosphatase-1 displays heterogeneity in isoelectric focusing, with a major band at pH 5.3. The enzyme hydrolyzes a wide variety of phosphate monoesters, including AMP, glucose 6-phosphate, ATP, choline phosphate, or phosphoproteins. The maximum reaction rates are different for all substrates, and some substrates appear to inhibit the reaction at high substrate concentrations. The Michaelis constants for 1-naphthyl phosphate and p-nitrophenyl phosphate are 79 microM and 68 microM, respectively, at pH 5.0 and 37 degrees C. The optimum pH level for 1-naphthyl phosphate is 4.5. Acid phosphatase-1 is inhibited by L(+)-tartrate (but not D(-)-tartrate), phosphate, and fluoride. The reaction rate increases 2.1-fold for every 10 degrees C rise in temperature. Above 48 degrees C, the rate of thermal denaturation is greater than the rate of the enzyme reaction.     

Modular organization of FDH: Exploring the basis of hydrolase catalysis

An abundant enzyme of liver cytosol, 10-formyltetrahydrofolate dehydrogenase (FDH), is an interesting example of a multidomain protein. It consists of two functionally unrelated domains, an aldehyde dehydrogenase-homologous domain and a folate-binding hydrolase domain, which are connected by an approximately 100-residue linker. The amino-terminal hydrolase domain of FDH (Nt-FDH) is a homolog of formyl transferase enzymes that utilize 10-formyl-THF as a formyl donor. Interestingly, the concerted action of all three domains of FDH produces a new catalytic activity, NADP+-dependent oxidation of 10-formyltetrahydrofolate (10-formyl-THF) to THF and CO2. The present studies had two objectives: First, to explore the modular organization of FDH through the production of hybrid enzymes by domain replacement with methionyl-tRNA formyltransferase (FMT), an enzyme homologous to the hydrolase domain of FDH. The second was to explore the molecular basis for the distinct catalytic mechanisms of Nt-FDH and related 10-formyl-THF utilizing enzymes. Our studies revealed that FMT cannot substitute for the hydrolase domain of FDH in order to catalyze the dehydrogenase reaction. It is apparently due to inability of FMT to catalyze the hydrolysis of 10-formyl-THF in the absence of the cosubstrate of the transferase reaction despite the high similarity of the catalytic centers of the two enzymes. Our results further imply that Ile in place of Asn in the FDH hydrolase catalytic center is an important determinant for hydrolase catalysis as opposed to transferase catalysis.     

Evaluation of substrate promiscuity of an L‐carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43

N‐carbamoyl‐amino‐acid amidohydrolase (also known as N‐carbamoylase) is the stereospecific enzyme responsible for the chirality of the D ‐ or L ‐amino acid obtained in the “Hydantoinase Process.” This process is based on the dynamic kinetic resolution of D ,L ‐5‐monosubstituted hydantoins. In this work, we have demonstrated the capability of a recombinant L ‐N‐carbamoylase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (BsLcar) to hydrolyze N‐acetyl and N‐formyl‐L ‐amino acids as well as the known N‐carbamoyl‐L ‐amino acids, thus proving its substrate promiscuity. BsLcar showed faster hydrolysis for N‐formyl‐L ‐amino acids than for N‐carbamoyl and N‐acetyl‐L ‐derivatives, with a catalytic efficiency (k_cat/K_m) of 8.58 × 10⁵, 1.83 × 10⁴, and 1.78 × 10³ (s^?1 M^?1), respectively, for the three precursors of L ‐methionine. Optimum reaction conditions for BsLcar, using the three N‐substituted‐L ‐methionine substrates, were 65°C and pH 7.5. In all three cases, the metal ions Co²⁺, Mn²⁺, and Ni²⁺ greatly enhanced BsLcar activity, whereas metal‐chelating agents inhibited it, showing that BsLcar is a metalloenzyme. The Co²⁺‐dependent activity profile of the enzyme showed no detectable inhibition at high metal ion concentrations. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Properties of a dolichol phosphokinase activity associated with rat liver microsomes

Rat liver microsomes show a capacity to synthesize [1-3H]dolichyl phosphate from [1-3H]-dolichol. Formation of [1-3H]dolichyl phosphate increased continuously over 15 min although the reaction rate was never completely linear. Product formation was directly proportional to microsomal protein concentration between 1.1 mg/mL and the highest concentration tested, 5.5 mg/mL. The reaction rate was linear with respect to the dolichol content of the assay mixture to a saturation point (120 microM). An apparent Km of 50 microM was established for dolichol. The normal phosphate donor for the reaction is CTP and not ATP. The optimum concentration of CTP was 10 mM, and an apparent Km of 4 mM was calculated for this nucleoside triphosphate. The reaction was totally dependent on divalent metal ion, magnesium being more effective than calcium. The optimum concentration of magnesium ion and CTP were the same (10 mM), suggesting that MgCTP2- is utilized as the normal enzyme substrate. Activity measured in the absence of Triton X-100 was only 5% of the activity observed at the optimum (0.5% w/v) detergent concentration. The measurable levels of dolichol phosphokinase could be doubled by the inclusion of 10-15 mM NaF as phosphatase inhibitor. Optimal enzymatic activity was obtained between pH 7.0 and pH 7.5 and could be inhibited by EDTA. The sulfhydryl reagent DTT was slightly stimulatory while the product of the reaction, dolichyl phosphate, was noninhibitory at the highest concentration tested (13.8 microM). The second reaction product (CDP) inhibits the enzymatic phosphorylation of dolichol.     

sn-Glycero(3)phosphoinositol glycerophosphohydrolase. A new phosphodiesterase in rat tissues.

1. A phosphodiesterase that cleaves glycerophosphoinositol into glycerophosphate and inositol has been detected in rat tissues. 2. The enzyme requires Mg2+ (Mn2+) and has a pH optimum of 7.7. 3. The richest sources of the enzyme are kidney and intestinal mucosa. In pancreas subcellular fractions it occurs largely in the microsomal fraction. 4. The enzyme is inhibited by excess substrate and by the reaction product glycerophosphate. 5. Temperature-stability studies and other observations distinguish the enzyme from other membrane-bound phosphodiesterases active at an alkaline pH e.g. glycerophosphoinositol inositophosphohydrolase, glycerophosphocholine diesterase, inositol cyclic phosphate phosphodiesterase and phosphodiesterase I.     

The role of formylmethanofuran: tetrahydromethanopterin formyltransferase in methanogenesis from carbon dioxide

Formylmethanofuran: tetrahydromethanopterin formyltransferase was purified to electrophoretic homogeneity from cells of Methanobacterium thermoautotrophicum. The enzyme is a tetramer of similar or identical subunits (Mr = 41,000). The equilibrium favors transfer of the formyl group to tetrahydromethanopterin (H4MPT) at physiological pH. The product of formyl transfer by the purified enzyme was shown by a number of criteria to be 5-formyl-H4MPT, as opposed to 10-formyl-H4MPT or 5,10-methenyl-H4MPT. Reconstitution of a portion of the methanogenic C1 cycle was effected by combining purified formyltransferase, methenyl-H4MPT cyclohydrolase, formylmethanofuran, and H4MPT to give methenyl-H4MPT. Additional reconstitution experiments established that the formyltransferase is an essential enzyme for the conversion of carbon dioxide to methane. In conjunction with previously published data (Donnelly, M.I., Escalante-Semerena, J.C., Rinehart, K. L., Jr., and Wolfe, R.S. (1985) Arch. Biochem. Biophys. 242, 430-439), these data substantiate the role of 5-formyl-H4MPT as an intermediate of methanogenesis.     

Biosynthesis of dolichyl diphosphate N-acetylglucosamine intermediates in Ascaridia galli microsomes.

1. N-acetylglucosamine-1-phosphate transferase was demonstrated in the microsomal fraction of Ascaridia galli. 2. The transferase reaction depends on exogenous dolichyl phosphate as lipid acceptor and was found to be inhibited by tunicamycin. 3. The enzyme activity was optimal in the presence of sodium deoxycholate as detergent and Mg cations after 10 min of incubation. 4. The product of the transferase reaction--dolichyl diphosphate N-acetylglucosamine was converted into lipid-disaccharide-dolichyl diphosphate N,N'-diacetylchitobiose. 5. The maximum level of the conversion was achieved at 5 mM concentration of unlabelled UDP-N-acetylglucosamine, while this conversion was negligible at lower UDP-N-acetylglucosamine concentrations (0.1 and 0.5 mM).     

New aspects of formation of 1,2-cyclic phosphates by phospholipase C-delta1

Phosphoinositide-specific phospholipase C-delta1 (PI-PLC-delta1) cleaves phosphatidylinositol 4,5-bisphosphate (PI-4,5-P(2), 1), 5-phosphate (PI-5-P, 2) and 4-phosphate (PI-4-P, 3) to form the mixture of the corresponding 4,5-, 5- and 4-phosphorylated inositol 1,2-cyclic phosphate (IcP) and 1-phosphate (IP) (4-6 and 7-9, respectively). In this work, we have studied the rates of the cleavage and the ratios of the cyclic-to-acyclic phosphate products under various pH and Ca(2+) concentration conditions using 31P NMR to monitor the reactions. In agreement with the previous report (Kim et al. Biochim. Biophys. Acta 1989, 163, 177), our results indicate that the IcP/IP ratios strongly depend on the reaction conditions, with the cyclic phosphate products formed predominantly at low pH (pH 5.0) and high calcium concentration (5 mM). Surprisingly, however, we have found that at pH 8.0 and 5 mM Ca(2+), PI-5-P rather than PI-4,5-P(2) is the most preferred substrate with the highest V(max). The cleavage of PI-5-P generated also more cyclic phosphate product than the other two substrates. In addition, we have studied the analogous reaction of phosphorothioate analogues of 1 with the sulfur placed in the nonbridging (10) or bridging (13) positions. We have found that the phosphorothioate analogue 10 produced exclusively the cyclic product 11, whereas the analogue 13 afforded exlusively the acyclic product 7. These results are discussed in terms of the mechanism of PI-PLC, where the cyclic product is formed by 'leaking' from the active site before its subsequent hydrolysis. The potential significance of the cyclic products in the signaling pathways is also discussed.     

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.