aminomethyltransferase and 5-10-methylenetetrahydrofolic-acid

One-carbon metabolism in yeast is an essential process that relies on at least one of three one-carbon donor molecules: serine, glycine, or formate. By a combination of genetics and biochemistry we have shown how cells regulate the balance of one-carbon flow between the donors by regulating cytoplasmic serine hydroxymethyltransferase activity in a side reaction occurring in the presence of excess glycine. This control governs the level of 5,10-methylene tetrahydrofolate (5,10-CH(2)-H(4)folate) in the cytoplasm, which has a direct role in signaling transcriptional control of the expression of key genes, particularly those encoding the unique components of the glycine decarboxylase complex (GCV1, GCV2, and GCV3). Based on these and other observations, we propose a model for how cells balance the need to supplement their one-carbon pools when charged folates are limiting or when glycine is in excess. We also propose that under normal conditions, cytoplasmic 5,10-CH(2)-H(4)folate is mainly directed to generating methyl groups via methionine, whereas one-carbon units generated from glycine in mitochondria are more directed to purine biosynthesis. When glycine is in excess, 5, 10-CH(2)-H(4)folate is decreased, and the regulation loop shifts the balance of generation of one-carbon units into the mitochondrion.

Topics: Adenine; Amino Acid Oxidoreductases; Aminohydrolases; Aminomethyltransferase; Animals; beta-Galactosidase; Carbon; Carrier Proteins; Choline; Cytoplasm; Dose-Response Relationship, Drug; Formate-Tetrahydrofolate Ligase; Fungal Proteins; Gene Expression Regulation, Fungal; Glycine; Glycine Decarboxylase Complex; Glycine Dehydrogenase (Decarboxylating); Glycine Hydroxymethyltransferase; Kinetics; Magnetic Resonance Spectroscopy; Methylenetetrahydrofolate Dehydrogenase (NADP); Mice; Mice, Knockout; Mitochondria; Mitochondrial Proteins; Models, Biological; Multienzyme Complexes; Plasmids; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Serine; Signal Transduction; Tetrahydrofolates; Transcription, Genetic; Transferases; Up-Regulation

High-molecular-mass proteins from pea (Pisum sativum) mitochondrial matrix retained on an XM-300 Diaflo membrane ('matrix extract') exhibited high rates of glycine oxidation in the presence of NAD+ and tetrahydropteroyl-L-glutamic acid (H4 folate) as long as the medium exhibited a low ionic strength. Serine hydroxymethyltransferase (SHMT) (4 x 53 kDa) and the four proteins of the glycine-cleavage system, including a pyridoxal phosphate-containing enzyme ('P-protein'; 2 x 97 kDa), a carrier protein containing covalently bound lipoic acid ('H-protein'; 15.5 kDa), a protein exhibiting lipoamide dehydrogenase activity ('L-protein'; 2 x 61 kDa) and an H4 folate-dependent enzyme ('T-protein'; 45 kDa) have been purified to apparent homogeneity from the matrix extract by using gel filtration, ion-exchange and phenyl-Superose fast protein liquid chromatography. Gel filtration on Sephacryl S-300 in the presence of 50 mM-KCl proved to be the key step in disrupting this complex. During the course of glycine oxidation catalysed by the matrix extract a steady-state equilibrium in the production and utilization of 5,10-methylene-H4 folate was reached, suggesting that glycine cleavage and SHMT are linked together via a soluble pool of H4 folate. The rate of glycine oxidation catalysed by the matrix extract was sensitive to the NADH/NAD+ molar ratios, because NADH competitively inhibited the reaction catalysed by lipoamide dehydrogenase.

Topics: Amino Acid Oxidoreductases; Aminomethyltransferase; Carrier Proteins; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Glycine; Glycine Decarboxylase Complex H-Protein; Glycine Dehydrogenase (Decarboxylating); Glycine Hydroxymethyltransferase; Hydroxymethyl and Formyl Transferases; Mitochondria; Multienzyme Complexes; NAD; Oxidation-Reduction; Plant Proteins; Plants; Pyruvate Dehydrogenase Complex; Tetrahydrofolates; Thioctic Acid; Transferases

T-protein, one of the components of the glycine cleavage system, catalyzes the synthesis of the H-protein-bound intermediate from methylenetetrahydrofolate, ammonia, and H-protein having a reduced lipoyl prosthetic group (Okamura-Ikeda, K., Fujiwara, K., and Motokawa, Y. (1982) J. Biol. Chem. 257, 135-139). Spectroscopic studies indicated that the utilization of methylenetetrahydrofolate occurred only in the presence of the three substrates, indicating the formation of a quaternary complex. The amount of methylenetetrahydrofolate consumed was equal to that of methylene carbon attached to H-protein. Steady-state kinetic studies show that the reaction proceeds through an Ordered Ter Bi mechanism. Reduced H-protein is the first substrate that binds T-protein followed by methylenetetrahydrofolate and ammonia. The order of release of products is tetrahydrofolate and the H-protein-bound intermediate. Km values for H-protein, methylenetetrahydrofolate, and ammonia are 0.55 microM, 0.32 mM, and 22 mM, respectively.

Topics: Amino Acid Oxidoreductases; Aminomethyltransferase; Ammonia; Ammonium Chloride; Carrier Proteins; Glycine Decarboxylase Complex H-Protein; Glycine Dehydrogenase (Decarboxylating); Hydroxymethyl and Formyl Transferases; Kinetics; Protein Binding; Tetrahydrofolates; Transferases

Glycine is converted to carbon dioxide and an intermediate attached to H-protein in the P-protein-catalyzed partial reaction of the glycine cleavage reaction (Fujiwara, K., and Motokawa, Y. (1983) J. Biol. Chem. 258, 8156-8162). The studies presented in this communication indicate that the amino group of glycine is retained in the intermediate and released as ammonia in the second partial reaction catalyzed by T-protein. The formation of ammonia accompanies the stoichiometric formation of 5,10-methylenetetrahydrofolate from the methylene carbon of glycine and tetrahydrofolate. Kinetic studies show that the reaction proceeds through a sequential mechanism. Km values for the intermediate complex and tetrahydrofolate are 2.2 and 50 microM, respectively. In the absence of tetrahydrofolate, T-protein catalyzes the stoichiometric formation of ammonia and formaldehyde from the intermediate although the velocity is extremely low. Km value for the intermediate complex in the absence of tetrahydrofolate is 10.3 microM, about 4-fold higher than the value in the presence of tetrahydrofolate. The addition of tetrahydrofolate increased the rate about 2400-fold. The modification of the free lipoyl sulfhydryl group with N-ethylmaleimide caused the intermediate complex inactive. The lipoyl sulfhydryl group seems to be essential for both P-protein- and T-protein-catalyzed partial reactions.

Topics: Amino Acid Oxidoreductases; Aminomethyltransferase; Ammonia; Animals; Carrier Proteins; Chickens; Glycine Decarboxylase Complex H-Protein; Glycine Dehydrogenase (Decarboxylating); Hydrogen-Ion Concentration; Hydroxymethyl and Formyl Transferases; Kinetics; Liver; Macromolecular Substances; Multienzyme Complexes; Tetrahydrofolates; Transferases

An inherited defect in the glycine cleavage enzyme results in the condition of neonatal glycine encephalopathy which has not responded to the current innovative methods of therapy. A re-examination of the enzyme structure and metabolic pathways, leads us to recommend future clinical evaluation of (1) vitamin-responsiveness, e.g., pyridoxine, folate and lipoic acid, (2) methionine, (3) N5, N10-methylene tetrahydrofolate and (4) alpha-methylserine therapy during the critical period of neonatal brain growth and development.

Topics: Amino Acid Metabolism, Inborn Errors; Amino Acid Oxidoreductases; Aminomethyltransferase; Animals; Brain Diseases, Metabolic; Carrier Proteins; Chemical Phenomena; Chemistry; Glycine; Glycine Dehydrogenase (Decarboxylating); Humans; Hydroxymethyl and Formyl Transferases; Infant; Infant, Newborn; Methionine; Mice; Serine; Tetrahydrofolates; Transferases