Inhibition of glycine N-methyltransferase by 5-methyltetrahydrofolate pentaglutamate

Glycine N-methyltransferase (EC 2.1.1.20) catalyzes the methylation of glycine by S-adenosylmethionine to form sarcosine and S-adenosylhomocysteine. The enzyme was previously shown to be abundant in both the liver and pancreas of the rat, to consist of four identical monomers, and to contain tightly bound folate polyglutamates in vivo. We now report that the inhibition of glycine N-methyltransferase by (6S)-5-CH(3)-H(4)PteGlu(5) is noncompetitive with regard to both S-adenosylmethionine and glycine. The enzyme exhibits strong positive cooperativity with respect to S-adenosylmethionine. Cooperativity increases with increasing concentrations of 5-CH(3)-H(4)PteGlu(5) and is greater at physiological pH than at pH 9.0, the pH optimum. Under the same conditions, cooperativity is much greater for the pancreatic form of the enzyme. The V(max) for the liver form of the enzyme is approximately twice that of the pancreatic enzyme, while K(m) values for each substrate are similar in the liver and pancreatic enzymes. For the liver enzyme, at pH 7.0 half-maximal inhibition is seen at a concentration of about 0.2 microM (6S)-5-CH(3)-H(4)PteGlu(5), while at pH 9.0 this value is increased to about 1 microM. For the liver form of the enzyme, 50% inhibition with respect to S-adenosylmethionine at pH 7.4 occurs at about 0.27 microM. The dissociation constant, K(s), obtained from binding data at pH 7.4 is 0.095. About 1 mol of (6S)-5-CH(3)-H(4)PteGlu(5) was bound per tetramer at pH 7.0, and 1.6 mol were bound at pH 9.0. The degree of binding and inhibition were closely parallel at each pH. At equal concentrations of (6R,6S)- and (6S)-5-CH(3)-H(4)PteGlu(5), the natural (6S) form was about twice as inhibitory. These studies indicate that glycine N-methyltransferase is a highly allosteric enzyme, which is consistent with its role as a regulator of methyl group metabolism in both the liver and the pancreas.     

Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.     

Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.     

Purification and properties of glycine N-methyltransferase from rat liver

Glycine N-methyltransferase (EC 2.1.1.20) has been purified to homogeneity from rat liver. The enzyme has a molecular weight of 132,000 by sedimentation equilibrium method. This value is in good agreement with a value of 130,000 obtained by Sephadex G-150 chromatography. The molecular weight of the denatured enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is 31,500. The numbers of peptides obtained by tryptic digestion and by cyanogen bromide cleavage are one-fourth of those expected from the contents of lysine plus arginine residues and methionine residues, respectively. By Edman degradation, phenylthiohydantoin-leucine is the only amino acid derivative released from the enzyme. Neither sugar nor phospholipid is detected in the purified preparation. These data indicate that the rat liver glycine N-methyltransferase is a simple protein consisting of 4 identical subunits. The enzyme has an isoelectric pH of 6.4, and is most active at pH 9.0. From the circular dichroism spectrum, an alpha helix content of about 11% is calculated. Whereas the initial velocity as a function of glycine concentration gives a Michaelis-Menten kinetics, the enzyme shows a positive cooperativity with respect to S-adenosylmethionine. The concentrations of glycine and S-adenosylmethionine which give a half-maximum velocity are 0.13 mM and 30 microM, respectively, at pH 7.4 and 25 degrees C.     

Identification of phosphorylation sites in glycine N-methyltransferase from rat liver

Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo, and could be phosphorylated in vitro on serine residues with a significant increase of enzyme activity, but no phosphorylation sites were identified. In this work the identification of the specific phosphorylation sites of rat GNMT is reported. Three different preparations of rat GNMT were analyzed: (1) purified from liver by standard methods of protein purification, (2) prepared from isolated hepatocytes and from liver tissue by immunoprecipitation, and (3) recombinant protein expressed in Escherichia coli. We measured the molecular weights of protein isoforms using electrospray mass spectrometry and used liquid chromatography-tandem mass spectrometry (LC-MS/MS) of peptides resulting from tryptic and chymotryptic digests. We also performed chemical analysis of phosphoamino acids and protein sequencing. In all samples, the phosphorylated serine residues 71, 182, and 241 were found. In GNMT prepared from liver tissue and hepatocytes an S9 additional residue was found to be phosphorylated. In hepatocytes and in recombinant GNMT S139 was detected. Serine 9 was also identified as a target for cAMP-dependent protein kinase in vitro. The positions of these phosphorylated residues in the tertiary structure of GNMT indicate their possible effect on enzyme conformation and activity.     

Characterization of glycine N-methyltransferase from rabbit liver.

The enzymatic properties of glycine N-methyltransferase from rabbit liver and the effects of endogenous adenosine nucleosides, nucleotides and methyltransferase inhibitors were investigated using a photometrical assay to detect sarcosine with o-dianisidine as a dye. After isolation and purification the denatured enzyme showed a two-banded pattern by SDS-PAGE. The enzyme was highly specific for its substrates with a pH-optimum at pH 8.6. Glycine N-methyltransferase exhibits Michaelis-Menten kinetics for its substrates, S-adenosylmethionine and glycine, respectively. The apparent Km and Vmax values were determined for both the substrates, the other substrate being present at saturating concentrations. The enzyme was strongly inhibited in the presence of S-adenosylhomocysteine, 3-deazaadenosine, and 5'-S-isobutylthio-5'-deoxyadenosine. All other inhibitors investigated, adenosine, 2'-deoxyadenosine, aciclovir, and 5'-N-ethylcarboxamidoadenosine were poor inhibitors of the methylation reaction. Adenine nucleotides and vidarabin were without effect on the enzymatic activity. Based on the kinetic data glycine N-methyltransferase from rabbit liver exhibits appreciable activity at physiological S-adenosylmethionine and S-adenosylhomocysteine levels.     

Studies of the role of a particulate folate-binding protein in the uptake of 5-methyltetrahydrofolate by cultured human KB cells

The characteristics of the uptake by human epidermoid carcinoma (KB) cells of 5-methyltetrahydrofolate at extracellular concentrations in the physiologic range and the possible role of a membrane-associated folate binder in folate uptake by KB cells have been investigated. Uptake of 5-methyltetrahydrofolate was specific, saturable, and time-, temperature-, and concentration-dependent. Trypsin treatment released 50% of the 5-methyltetrahydrofolate accumulated by KB cells at 4 degrees C, but only 12% at 37 degrees C, indicating that most of the accumulated ligand was intracellular at 37 degrees C, thus demonstrating transport. Accumulated 5-methyltetrahydrofolate was bound to a membrane-associated protein which required detergent for its solubilization, and a significant amount of which was oriented to the cell exterior as demonstrated by its release by trypsin treatment of intact KB cells. The membrane-associated folate binder was immunoprecipitated by antiserum to purified human placental folate receptor, and this antiserum inhibited 5-methyltetrahydrofolate uptake by intact KB cells in a concentration-dependent manner. These data support the hypothesis that the membrane-associated folate-binding protein of human cells participates in the transport of folates under physiologic conditions.     

Reaction of rat liver glycine methyltransferase with 5'-p-fluorosulfonylbenzoyladenosine

Rat liver glycine methyltransferase is inactivated by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) in a pseudo-first order fashion at pH 7.5. The addition of dithiothreitol (20 mM) to the reaction mixture results in partial restoration of enzyme activity. A semilog plot of residual activity after dithiothreitol reactivation versus time is also linear, indicating that at least two essential residues are present on the enzyme and the modification of either of which causes total loss of activity. The inactivation is accompanied by incorporation of the radiolabel from adenine-labeled FSBA, but the amount of radioactivity fixed is not altered upon treatment with dithiothreitol. From this fact and the stoichiometry between the loss of dithiothreitol-sensitive activity and the number of sulfhydryl groups disappeared, it is suggested that the dithiothreitol-sensitive inactivation is the consequence of the FSBA-mediated formation of a disulfide between two sulfhydryl groups in close proximity. Although 4 mol of reagent are covalently bound per enzyme subunit, the kinetics of modification and inactivation show that the reaction at 1 residue, which is identified as tyrosine, is responsible for the dithiothreitol-insensitive inactivation. The substrate S-adenosylmethionine provides complete protection against both types of inactivation, but the dithiothreitol-insensitive inactivation is protected much more effectively with a Kd value comparable to the Km value. This suggests that the tyrosine is located at or near the active site of the methyltransferase.     

A protease is bound to rat liver nucleosomes

Nucleosome prepared from pure rat liver nuclei contain protease activity. No protease is associated with nucleosome core particle and the protease-containing nucleosome has considerably higher sedimentation coefficient than the bulk nucleosomes. Only H1 histone is susceptible to the nucleosome-bound protease.     

Purification of phosphatidylethanolamine N-methyltransferase from rat liver

Phosphatidylethanolamine (PE) N-methyltransferase catalyzes the synthesis of phosphatidylcholine by the stepwise transfer of methyl groups from S-adenosylmethionine to the amino head group of PE. PE N-methyltransferase was solubilized from a microsomal membrane fraction of rat liver using the nonionic detergent Triton X-100 and purified to apparent homogeneity. Specific activities of PE N-methyltransferase with PE, phosphatidyl-N-monomethylethanolamine (PMME), and phosphatidyl-N,N-dimethylethanolamine (PDME) as substrates were 0.63, 8.59, and 3.75 mumol/min/mg protein, respectively. The purified enzyme was composed of a single subunit with a molecular mass of 18.3 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methylation activities dependent on the presence of PE, PMME, and PDME and the 18.3-kDa protein co-eluted when purified PE N-methyltransferase was subjected to gel filtration on Sephacryl S-300 in the presence of 0.1% Triton X-100. All three methylation activities eluted with a Stokes radius 2.1 A greater than that determined for pure Triton micelles (molecular mass difference of 27.4 kDa). Two-dimensional analysis of PE N-methyltransferase employing nonequilibrium pH gradient gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of a single isoform. Analysis of enzyme activity using PE, PMME, and PDME at various Triton X-100 concentrations indicated the enzyme follows the "surface dilution" model proposed for other enzymes that act at the surface of mixed micelle substrates. Initial velocity data for all three lipid substrates (at fixed concentrations of Triton X-100) were highly cooperative in nature. Hill numbers for PMME and PDME ranged from 3 at 0.5 mM Triton to 6 at 2.0 mM Triton. All three methylation activities had a pH optimum of 10. These results provide evidence that a single membrane-bound enzyme catalyzes all three methylation steps for the conversion of PE to phosphatidylcholine.     

Determination of mouse liver 5-methyltetrahydrofolate concentration and polyglutamate forms

The concentration and polyglutamate status of 5-methyltetrahydrofolate in mouse liver tissue extracts has been determined by enzymatic conversion to methylenetetrahydrofolate and subsequent entrapment of this confactor form into a ternary complex with Lactobacillus casei thymidylate synthase and tritiated 5-fluorodexyuridylate. 5-Methyltetrahydrofolate was oxidized to methylenetetrahydrofolate using the reverse reaction of methylenetrahydrofolate reductase with menadione as the ultimate electron acceptor. Reference 5-methylterahydrofolate could be quantitatively recovered from tissue extracts by this method. The polyglutamate status of enzymatically converted and complexed tissue 5-methyltetrahydrofolate was determine electrophoretically. Unlabeled 5-fluorodeoxyuridylate was used to remove endogenous methylenetetrahydrofolate prior to enzymatic oxidation of 5-methyltetrahydrofolate and subsequent electrophoretic analysis. In this manner, the 5-methyltetrahydrofolate polyglutamate pool alone could be labeled and visualized. There were no observable differences in the polyglumate distribution of endogenous methylenetetrahydrofolate versus 5-methyltetrahydrofolate polyglutamates of normal mouse liver tissue.     

Presence of di- and polyamines covalently bound to protein in rat liver

Acid hydrolysis of trichloroacetic acid precipitate from rat tissue (liver, kidney and testis) homogenate released significant amounts of acid-insoluble putrescine, spermidine and spermine. Following incubation of liver homogenate with [1,4-¹⁴C]putrescine, 1.4% of total radioactivity and 1.0% of labelled diamine were recovered in the acid-insoluble fraction. Exhaustive digestion of acid-precipitable material with proteinases (Pronase, aminopeptidase M, carboxipeptidase A, B and Y) revealed the presence of di- and polyamines and of N¹-(γ-glutamyl)spermidine, N¹-(γ-glutamyl)sperminine and N¹, N¹²-bis(γ-glutamyl)spermine. These derivatives were identified both by chromatographic analysis and by enzymatic digestion with purified γ-glutamylamine cyclotransferase. The finding of di- and polyamine γ-glutamyl derivatives in the proteinase-digested acid-insoluble fraction of homogenate may be considered as a proof of the in vivo transglutaminase-catalyzed binding of polyamines to proteins. This evidence suggests that di- and polyamines might have an important role in mammalian tissues through covalent binding to proteins by either one or both the primary amino groups.     

Distribution of bound ADP-ribose derivatives in nuclear protein of rat liver nuclei

Most of the acid-insoluble radioactivity produced by incubation of rat liver nuclei with [¹⁴C]NAD is rendered soluble by treatment with cold neutral hydroxylamine. The substances released by hydroxylamine have been determined to be (adenosine diphosphoribose)oligomer, adenosine diphosphoribose, 5′-AMP and adenosine, the greatest activity being found in the adenosine diphosphoribose fraction. The distribution of hydroxylamine-sensitive radioactive material in the nuclear proteins varies with the fractionation method employed. Regardless of the method employed, the “histones” contained only small amounts of hydroxylamine-insensitive radioactive material [poly(adenosine diphosphoribose)].     

Characterization of an arginine-specific protein kinase tightly bound to rat liver DNA

A new protein kinase has been characterized among the proteins tightly bound to rat liver DNA and released by DNase I and RNase A treatment. This enzyme was separated by gel filtration from this released material. Its apparent molecular mass was found to be 34 kDa and it is made of a single unit. The main characteristic of this protein kinase is that it is arginine-specific. Isolation of phosphoarginine required the use of proteolytic enzymes at alkaline pH since the phosphate bond is highly acid-labile. This protein kinase is able to autophosphorylate and to phosphorylate a single chromosomal protein of 11 kDa also tightly bound to DNA. It uses ATP and dATP as phosphate donors and is cAMP-independent. Its optimal activity requires Mn2+ ions. Vanadate, spermine and heparin have no effect on its activity.