Mechanism of the glycine cleavage reaction: retention of C-2 hydrogens of glycine on the intermediate attached to H-protein and evidence for the inability of serine hydroxymethyltransferase to catalyze the glycine decarboxylation

Glycine is converted to carbon dioxide and an intermediate attached to a lipoic acid group on H-protein in the P-protein-catalyzed partial reaction of the glycine cleavage reaction [K. Fujiwara and Y. Motokawa (1983) J. Biol. Chem. 258, 8156-8162]. The results presented in this paper indicate that the decarboxylation is not accompanied by the removal of a C-2 hydrogen atom of glycine and instead both C-2 hydrogens are transferred with the alpha carbon atom to the intermediate formed during the decarboxylation of glycine. The purified chicken liver cytosolic and mitochondrial serine hydroxymethyltransferase preparations could not catalyze the decarboxylation of glycine in the presence of either lipoic acid or H-protein. The decarboxylation activity of the serine hydroxymethyltransferase preparation purified from bovine liver by the method similar to that of L. R. Zieske and L. Davis [(1983) J. Biol. Chem. 258, 10355-10359] was completely inhibited by the antibody to P-protein, while the antibody had no effect on the activity of the phenylserine cleavage. Conversely, D-serine inhibited the activity of phenylserine cleavage but the activity of the decarboxylation of glycine was not affected by D-serine. Finally, the two activities were separated by the chromatography on hydroxylapatite. The results clearly demonstrate that serine hydroxymethyltransferase per se cannot catalyze the decarboxylation of glycine.     

Glycine Serine Interconversion in the Rooster

Serine was isolated by the column chromatography from the hydrolyzates of proteins of the serum, the liver and the pectoral muscle which were obtained from the roosters fed a diet containing 2-¹⁴C glycine for 16–17 days. The carbon chain of serine was cut off by treating with sodium periodate. The specific activity of each carbon (as barium carbonate) was estimated. Carboxyl carbon had little radioactivity. The specific activity of hydroxymethyl carbon was 10–19% of that of methylen carbon. Glycine isolated from the same hydrolyzates was degraded by ninhydrin oxidation. Formaldehyde produced from 2-C was oxidized to carbon dioxide by treating with mercuric chloride. Carboxyl carbon had little radioactivity. The specific activities of 2-C of glycine and 2-C of serine in the same tissue protein were compared. The ratio of serine 2-C/glycine 2-C was between 0.7 – 1.5. These results seem to indicate that glycine directly converts to serine in the rooster. The quantitative significance of the pathways of glycine (serine) biosynthesis is discussed.     

Correlation between the serine sensitivity and the derepressibility of the ilv genes in Escherichia coli relA- mutants.

Summary Upon addition of excess one carbon metabolites (including serine) bacteria stop growing because of isoleucine starvation. After such treatment stringent bacteria rapidly resume normal growth whereas relaxed mutants remain unable for some time to grow. We show here that this is due to a lack of derepressibility of ilv genes after the starvation period. Results are also presented which show that RNA polymerase structural mutants may be selected among the clones resistant to a mixture of serine, methionine and glycine, in relA ^-strains. Finally circumstancial evidence suggests that the one carbon metabolism may be involved in a process controlling isoleucine metabolism.Abbreviations: Throughout this work we have represented the mixture of amino acids serine, methionine and glycine (1 mM each) by the letters SMG. Para amino benzoic acid represented by the letters PABA     

In vitro poly(ADP-ribosyl)ation of seminal ribonuclease

The site of in vitro ADP-ribosylation of seminal ribonuclease was determined. Seminal enzyme was found to be a good receptor of [14C]ADP-ribose residues under the reaction conditions used. The recovery of [14C]ADP-ribosylated RNase was about 65% after purification. After tryptic digestion of modified enzyme, a fraction containing [14C]ADP-ribosylated peptides was separated from the others by ion-exchange chromatography on M82 resin. Radioactive peptides were then purified by affinity chromatography on anti-poly(ADP-ribose)IgG-Sepharose. High performance liquid chromatography of a mixture obtained after pronase digestion of purified ADP-ribosylated peptides revealed only one radioactive peptide whose amino acid composition corresponded to a peptide that has equimolar quantities of aspartic acid, serine, and glycine. Carboxypeptidase Y digestion of this peptide showed that its amino acid sequence was Asp-Ser-Gly. Only position 14-16 of seminal RNase corresponded to this sequence. The chemical stability of the ADP-ribose/enzyme linkage indicated that aspartic acid 14 is the modification site in seminal RNase.     

Glycine transport in mouse eggs and preimplantation embryos

In pre-compaction embryos glycine was taken up by the glycine-specific gly-system, which is concentrative, weakly exchangeable and dependent on Na+. After compaction glycine uptake increased, apparently due to the expression of the A-transport system and its reactivity with glycine. Studies of the metabolic fate of carbon from glycine indicated conversion to serine and alanine. These changes are interpreted to show that glycine could provide carbon for intermediary energy metabolism, resulting in CO2, as well as for macromolecular synthesis.     

Gluconeogenesis from glycine and serine in fasted normal and diabetic rats.

1. Non-anaesthetized normal and diabetic rats were fasted for 1 day, and [U-14C]glycine, or [U-14C]serine, or [U-14C]- plus [3-3H]-glucose was injected intra-arterially. The rates of synthesis de novo/irreversible disposal for glycine, serine and glucose, as well as the contribution of carbon atoms by the amino acids to plasma glucose, were calculated from the integrals of the specific-radioactivity-versus-time curves in plasma. 2. The concentrations of both glycine and serine in blood plasma were lower in diabetic than in fasted normal animals. 3. The rates of synthesis de novo/irreversible disposal of both amino acids tended to be lower in diabetic animals, but the decrease was statistically significant only for serine (14.3 compared with 10.5 mumol/min per kg). 4. Of the carbon atoms of plasma glucose, 2.9% arose from glycine in both fasted normal and diabetic rats, whereas 4.46% of glucose carbon originated from serine in fasted normal and 6.77% in diabetic rats. 5. As judged by their specific radioactivities, plasma serine and glycine exchange carbon atoms rapidly and extensively. 6. It was concluded that the turnover of glycine remains essentially unchanged, whereas that of serine is decreased in diabetic as compared with fasted normal rats. The plasma concentration of both amino acids was lower in diabetic rats. Both glycine and serine are glucogenic. In diabetic rats the contribution of carbon atoms from glycine to glucose increases in direct proportion to the increased glucose turnover, whereas the contribution by serine becomes also proportionally higher.     

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.     

Mechanism of acetate synthesis from CO2 by Clostridium acidiurici.

Total synthesis of acetate from CO2 by Clostridium acidiurici during fermentations of hypoxanthine has been shown to involve synthesis of glycine from methylenetetrahydrofolate, CO2, and NH3. The glycine is converted to serine by the addition of methylenetetrahydrofolate, and the resulting serine is converted to pyruvate, which is decarboxylated to form acetate. Since CO2 is converted to methylenetetrahydrofolate, both carbons of the acetate are derived from CO2. The evidence supporting this pathway is based on (i) the demonstration that glycine decarboxylase is present in C. acidiurici, (ii) the fact that glycine is synthesized by crude extracts at a rate which is rapid enough to account for the in vivo synthesis of acetate from CO2, (iii) the fact that methylenetetrahydrofolate is an intermediate in the formation of both carbons of acetate from CO2, and (iv) the fact that the alpha carbon of glycine is the source of the carboxyl group of acetate. Evidence is presented that this synthesis of acetate does not involve carboxylation of a methyl corrinoid enzyme such as occurs in Clostridium thermoaceticum and Clostridium formicoaceticum. Thus, there are two different mechanisms for the total synthesis of acetate from CO2 by clostridia.     

Evidence for the Involvement of Serine Transhydroxymethylase in Serine and Glycine Interconversions in SALMONELLA TYPHIMURIUM

Salmonella typhimurium can normally use glycine as a serine source to support the growth of serine auxotrophs. This reaction was presumed to occur by the reversible activity of the enzyme, serine transhydroxymethylase (E. C. 2. 1. 2. 1; L-serine: tetrahydrofolic-5, 10 transhydroxymethylase), which is responsible for glycine biosynthesis. However, this enzyme had not been demonstrated to be solely capable of synthesizing serine from glycine in vivo. The isolation and characterization of a mutant able to convert serine to glycine but unable to convert glycine to serine supports the conclusion that a single enzyme is involved in this reversible interconversion of serine and glycine. The mutation conferring this phenotype was mapped with other mutations affecting serine transhydroxymethylase (glyA) and assays demonstrated reduced activities of this enzyme in the mutant.     

Glycine metabolism by rat liver mitochondria. Reconstruction of the reversible glycine cleavage system with partially purified protein components

An enzyme system which catalyzes the degradation of glycine to one carbon unit, ammonia, and carbon dioxide and the synthesis of glycine from these three substances has been isolated from rat liver mitochondria. The reversible glycine cleavage system is composed of four protein components named as P-, H-, L-, and T-protein, respectively. A procedure is described for the purification of P-protein which catalyzes the decarboxylation of glycine or its reverse reaction in the presence of H-protein, and for T-protein which participates in the formation of one carbon unit and ammonia or the reverse reaction. The procedure described leads to the isolation of a nearly homogeneous form of T-protein but P-protein still is heterogeneous. The molecular weight of T-protein, estimated by molecular sieve chromatography, is 33,000. Properties of the synthesis and cleavage reactions and the exchange of carboxyl group of glycine with bicarbonate are also presented.     

Structural requirements for nucleophilic substrates of carboxypeptidase Y

The composition and structural aspects of the amino and carboxylic acid groups required for incorporation into peptides by transpeptidation and inhibition of hydrolysis in carboxypeptidase Y-catalyzed reactions were studied. Separation of these two groups by even one carbon prevents incorporation by transpeptidation and does not inhibit incorporation of other amino acids into model peptides. Substitution of phosphonic or sulfonic acids for the carboxylic acid group also results in loss of incorporation by transpeptidation. Only the sulfonic acid analog of glycine causes inhibition of hydrolysis and this inhibition is lost when serine is included in the reaction. d-Serine is not incorporated by carboxypeptidase Y, and its presence in the reaction mixture does not inhibit the incorporation of the L-isomer.     

Chemical evidence for the separation of G gamma and A gamma chains by polyacrylamide gel electrophoresis in urea and triton X-100

Polyacrylamide gel electrophoresis in urea and Triton X-100 of a hemolysate from human fetal red blood cells produces four major protein bands: alpha, beta, and 2 gamma globin chains. We have verified that the latter two are the G gamma and A gamma globin chains which have respectively glycine or alanine at position 136. After incorporation of either [3H] alanine or [3H] glycine into newly synthesized globin each gamma chain was isolated by preparative electrophoresis. The chains were cleaved with cyanogen bromide at methionines 55 and 133, then subjected to automated sequencing, and the residues from each sequencer turn counted. Glycine incorporation was detected for the third turn (position 136) of the G gamma chain and alanine for the A gamma. Substantial metabolic conversion of [3H] glycine to serine and proline was also noted.     

Luteinizing hormone of the sperm whale. Isolation, separation into subunits and study of the amino acid sequence of the alpha-subunit

The luteinizing hormone isolated from sperm-whale pituitary was separated into two subunits, alpha- and beta-, by ion-exchange chromatography on sulfoethyl-Sephadex. The hormone subunits were reconstituted, carboxymethylated and cleaved by BrCN and proteolytic enzymes. In order to block tryptic hydrolysis at lysine residues the alpha-subunit was subjected to maleylation. Large-sized fragments of BrCN were cleaved by chymotrypsin and trypsin, while large-sized fragments of trypsin were split by chymotrypsin. The resulting peptides were separated by gel filtration on Sephadex, ion-exchange chromatography on Aminex A-5 and thin-layer partition chromatography on cellulose. The amino acid sequence of the peptides was determined by the Edman method, using identification of the N-terminal amino acids in a reaction with dansyl chloride or dimethylaminoazobenzene-4-isothiocyanate. It was shown that the alpha-subunit of the luteinizing hormone is a peptide chain consisting of 96 amino acid residues with covalently linked carbon chains at asparagine residues at positions 56 and 82. The N-terminal amino acid of the alpha-subunit is phenylalanine, the C-terminal amino acid is serine. The alpha-subunit is heterogeneous at the N-end, i. e. beside phenylalanine it contains threonine and trace amounts of proline, aspartate, glutamate and glycine.     

Utilization of l-threonine by a species of Arthrobacter. A novel catabolic role for `aminoacetone synthase''

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, (14)C from l-[U-(14)C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U-(14)C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2-(14)C]acetate and [2-(14)C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ;serine pathway'. 4. The threonine-grown organism contained ;biosynthetic' threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the alpha-amino-beta-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an alpha-amino-beta-oxobutyrate CoA-ligase, which was identified with ;aminoacetone synthase'. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and alpha-amino-beta-oxobutyrate CoA-ligase (i.e. ;aminoacetone synthase'), l-[U-(14)C]threonine was broken down to [(14)C]glycine plus [(14)C]acetyl-CoA (trapped as [(14)C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.     

Hydroxy amino acid metabolism in Pseudomonas cepacia: role of L-serine deaminase in dissimilation of serine, glycine, and threonine.

Growth of Pseudomonas cepacia (P. multivorans) on serine depended upon induction of a previously undescribed L-serine deaminase distinct from threonine deaminase. Formation of the enzyme was induced during growth on serine, glycine, or threonine. The induction pattern reflected a role of the enzyme in catabolism of these three amino acids. Both threonine and glycine supported growth of serine auxotrophs and were presumably converted to serine and pyruvate in the course of their degradation. Mutant strains deficient in serine deaminase, or unable to use pyruvate as a carbon source, failed to utilize serine or glycine and grew poorly with threonine, whereas strains deficient in threonine dehydrogenase or alpha-amino beta-ketobutyrate:coenzyme A ligase (which together convert threonine to glycine and acetyl coenzyme A) failed to utilize threonine or derepress serine deaminase in the presence of this amino acid. The results confirm for the first time the role of alpha-amin beta-ketobutyrate:coenzyme A ligase in threonine degradation and indicate that threonine does not mimic serine as an inducer of serine deaminase.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

Amino acids derivatives synthesis from nitrogen, carbon and water by electric discharges

Electric discharges between a pair of carbon electrodes were continued for 50 days in a vessel of 5 liters in volume which initially contained nitrogen at a pressure of 15 cm Hg and 200 ml of water. The pressure in the vessel was gradually increased to 60 cm Hg at the end of the run. Gas chromatographic analysis showed that the increase of the pressure mainly results from the production of hydrogen and carbon monoxide. The concentration of ammonia in the aqueous sample was increased to 0.05 M in 50 days of the discharge. After hydrolysis, glycine and serine were detected at the concentrations of 3.4 x 10(-3) M and 0.057 x 10(-3) M in the final solution, respectively, though glycine was found only at the concentration of 6 x 10(-6) M before hydrolysis. TLC analysis indicated the presence of hydantoic acid, N-formylglycine, diketopiperazine, and polymers of glycine.     

Glycine and serine catabolism in non-photosynthetic higher plant cells: their role in C1 metabolism

Glycine and serine are two interconvertible amino acids that play an important role in C1 metabolism. Using 13C NMR and various 13C-labelled substrates, we studied the catabolism of each of these amino acids in non-photosynthetic sycamore cambial cells. On one hand, we observed a rapid glycine catabolism that involved glycine oxidation by the mitochondrial glycine decarboxylase (GDC) system. The methylenetetra- hydrofolate (CH2-THF) produced during this reaction did not equilibrate with the overall CH2-THF pool, but was almost totally recycled by the mitochondrial serine hydroxymethyltransferase (SHMT) for the synthesis of one serine from a second molecule of glycine. Glycine, in contrast to serine, was a poor source of C1 units for the synthesis of methionine. On the other hand, catabolism of serine was about three times lower than catabolism of glycine. Part of this catabolism presumably involved the glycolytic pathway. However, the largest part (about two-thirds) involved serine-to-glycine conversion by cytosolic SHMT, then glycine oxidation by GDC. The availability of cytosolic THF for the initial SHMT reaction is possibly the limiting factor of this catabolic pathway. These data support the view that serine catabolism in plants is essentially connected to C1 metabolism. The glycine formed during this process is rapidly oxidized by the mitochondrial GDC-SHMT enzymatic system, which is therefore required in all plant tissues.     

Photorespiratory nitrogen cycle – A critical evaluation

The photorespiratory nitrogen cycle proposed by Keys et al. (Nature 275: 741–743, 1978) involved formation of glycine by transamination of glyoxylate in the peroxisomes utilizing glutamate. Subsequently, glycine is oxidized to ammonia, serine and CO₂ in the mitochondria. The ammonia is reassimilated via the GS/GOGAT pathway generating glutamate. In this article, experimental evidence which suggests the occurrence of alternative mechanisms of glycolate and serine synthesis as well as of CO₂ and ammonia evolution is discussed. The problem of utilization of NADH coupled to ATP synthesis during photosynthesis is still unresolved, which complicates the glycine oxidation reaction in light. Further, factors are presented that determine the availability of amino donors in the peroxisomes and of amino acids viz., glycine, serine and glutamate for the operation of the photorespiratory N cycle. Recent evidence regarding the role of formate arising out of the reaction of glyoxylate with H₂O₂ in the regulation of photosynthetic electron flow in the Hill reaction, as well as of photorespiratory substrates functioning as carbon sources for the citric acid cycle in the light or for export to the growing tissues, suggests that the role of photo-respiration in plant metabolism needs to be reexamined.     

Decarboxylation of glycine by serine hydroxymethyltransferase in the presence of lipoic acid

Serine hydroxymethyltransferase and the glycine cleavage system are both present in liver mitochondria and both bind glycine to form a pyridoxal 5'-phosphate carbanionic quinoid species. Lipoic acid has been shown to have the ability to intercept the carbanionic intermediate formed from the binary complex of serine hydroxymethyltransferase and glycine and form an intermediate adduct which is ultimately processed to yield CO2 and a methylamine adduct. Kinetic studies have shown that the lipoic acid-dependent decarboxylation of glycine catalyzed by serine hydroxymethyltransferase proceeds through a sequential mechanism. This lipoic acid-dependent decarboxylation catalyzed by serine hydroxymethyltransferase is similar to the initial reaction of the glycine cleavage system and to the lipoic acid-dependent decarboxylation of glycine by the P-protein alone suggesting that both enzymes could serve in lieu of each other.