Mechanism and stereochemistry of the 5-aminolaevulinate synthetase reaction

1. Two mechanisms for the biosynthesis of 5-aminolaevulinate from glycine and succinyl-CoA (3-carboxypropionyl-CoA) are considered. One of the mechanisms involves the retention of both the C-2 H atoms of glycine during the synthesis of 5-aminolaevulinate, whereas the other predicts the retention of only one of the C-2 H atoms of glycine. 2. Highly purified 5-aminolaevulinate synthetase from Rhodopseudomonas spheroides was used to show that the C-2 H atom of glycine with R configuration is specifically removed during the biosynthesis of 5-aminolaevulinate. 3. The mechanism of the condensation therefore differs from the analogous reaction of the biosynthesis of sphinganine from palmitoyl-CoA and serine, in which the C-2 H of serine is retained (Wiess, 1963).     

Incorporation of [2-14C,(5R)-5-3H1]mevalonic acid into cholesterol by a rat liver homogenate and into β-sitosterol and 28-isofucosterol by Larix decidua leaves

1. Incubation of a rat liver homogenate with 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid gave cholesterol with (3)H/(14)C atomic ratio 6:5. 2. Conversion of the labelled cholesterol into 3beta-acetoxy-6-nitrocholest-5-ene or cholest-4-ene-3,6-dione resulted in the loss of one tritium atom from C-6. 3. These results show that during cholesterol biosynthesis the 6alpha-hydrogen atom of a precursor sterol is eliminated during formation of the C-5-C-6 double bond. 4. Incorporation of 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid into the sterols of larch (Larix decidua) leaves gave labelled cycloartenol and beta-sitosterol with (3)H/(14)C atomic ratios 6:6 and 6:5 respectively. 5. One tritium atom was lost from C-6 on conversion of the labelled beta-sitosterol into either 3beta-acetoxy-6-nitrostigmast-5-ene or stigmast-4-ene-3,6-dione, demonstrating that formation of the C-5-C-6 double bond of phytosterols also involves the elimination of the 6alpha-hydrogen atom of a precursor sterol. 6. The 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid was also incorporated by larch (L. decidua) leaves into a sterol that co-chromatographed with 28-isofucosterol. Confirmation that the radioactivity was associated with 28-isofucosterol was obtained by co-crystallization with carrier 28-isofucosterol and ozonolysis of the acetate to give radioactively labelled 24-oxocholesteryl acetate. 7. The significance of these results to phytosterol biosynthesis is discussed.     

The stereochemistry of hydrogen elimination from C-7 in cholesterol and ergosterol biosynthesis

The synthesis of [7alpha-(3)H]lanosterol is described. It is shown that in the conversion of [7alpha-(3)H,26,27-(14)C(2)]lanosterol into cholesterol by a rat liver system, it is the 7beta-hydrogen atom that is predominantly removed. On the other hand, the conversion of doubly labelled lanosterol into ergosterol by whole yeast cells results in the loss of the 7alpha-hydrogen atom. These results therefore suggest that the C-7 hydrogen atoms with opposite stereochemistry are labilized by the rat liver and the yeast Delta(8)-Delta(7) steroid isomerases.     

Sphingomyelin synthesis in Ascaridia galli

Adult Ascaridia galli incorporate label from [U-¹⁴C] serine into various intermediates of sphingomyelin synthesis (ketosphinganine, sphinganine, sphingosine, ceramide and sphingomyelin). From the results it is concluded that A. galli possesses the five enzymes involved in sphingomyelin synthesis, namely: serine palmitoyltransferase, 3-ketosphinganine reductase, flavoprotein sphinganine reductase, sphingosine acyltransferase and ceramide choline phosphotransferase.     

Biosynthesis of long-chain (sphingoid) bases from serine by LM cells. Evidence for introduction of the 4-trans-double bond after de novo biosynthesis of N-acylsphinganine(s)

The de novo biosynthesis of sphinganine and sphingosine was studied using LM cells incubated with [14C] serine in serum-free media. Most of the radiolabeled long-chain bases were initially found in dihydroceramides (as sphinganine) and the proportion appearing in complex sphingolipids (as sphingosine) increased over time. Since free long-chain bases were not detected (although formation of 3-ketosphinganine, the first condensation product of serine and palmitoyl-CoA, could be demonstrated in vitro), it appears that the first step is rate-limiting for dihydroceramide biosynthesis. The kinetics suggested that after N-acyl-sphinganines were formed they were dehydrogenated to N-acylsphingosines. No evidence was found for the formation in vivo or in vitro of the putative intermediates of the direct biosynthesis of sphingosine from sphinganine (i.e. 3-ketosphingosine and free sphingosine). The conversion of N-acylsphinganines to N-acyl-sphingosines was confirmed by incubating cells with [14C] serine followed by unlabeled serine, which resulted in a rapid increase in the sphingosine-to-sphinganine ratio in amide-linked sphingolipids during the chase. These findings are most consistent with a pathway for long-chain base biosynthesis in which N-acyl-sphinganines are first synthesized by LM cells and the 4-trans-double bond is added to this or subsequent products, as opposed to the most cited pathway wherein sphingosine is made directly from sphinganine.     

Enzymatic in situ determination of stereospecificity of NAD-dependent dehydrogenases

Amino acid racemases inherently catalyze the exchange of alpha-hydrogen of amino acids with deuterium during racemization in 2H2O. When the reactions catalyzed by alanine racemase (EC 5.1.1.1) and L-alanine dehydrogenase (EC 1.4.1.1), which is pro-R specific for the C-4 hydrogen transfer of NADH, are coupled in 2H2O, [4R-2H]NADH is exclusively produced. Similarly, [4S-2H]NADH is made in 2H2O with amino-acid racemase with low substrate specificity (EC 5.1.1.10) and L-leucine dehydrogenase (EC 1.4.1.9), which is pro-S specific. We have established a simple procedure for the in situ analysis of stereospecificity of C-4 hydrogen transfer of NADH by an NAD-dependent dehydrogenase by combination with either of the above two couples of enzymes in the same reaction mixture. When the C-4 hydrogen of NAD+ is fully retained after sufficient incubation, the stereospecificity of hydrogen transfer by a dehydrogenase is the same as that of alanine dehydrogenase or leucine dehydrogenase. However, when the C-4 hydrogen of NAD+ is exchanged with deuterium, the enzyme to be examined shows the different stereospecificity from alanine dehydrogenase or leucine dehydrogenase. Thus, we can readily determine the stereospecificity by 1H NMR measurement without isolation of the coenzymes and products.     

Studies on the metabolism of unsaturated fatty acids. XII. Reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli

Incorporation of deuterium atoms from deuterium-labeled NADPH and 2H2O during the reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli (E. coli) was investigated. When trans-2,cis-4-decadienoyl-CoA was incubated with 4R- or 4S-[4-2H1]NADPH in the presence of purified 2,4-dienoyl-CoA reductase, no deuterium was detected in the reaction product by gas chromatography-mass spectrometry after derivatization to its pyrrolidine amide. On the other hand, when the dienoyl-CoA was incubated in the presence of NADPH and the reductase in 2H2O, two deuterium atoms were incorporated: One deuterium atom was located at the C-4 position of trans-2-decenoate, and the other at the C-5 position. The UV and shorter wavelengths of the visible spectrum of the reductase solution revealed that the reductase contained flavin as a prosthetic group. Therefore it is considered that a hydrogen atom of NADPH was first transferred to the flavin moiety of the reductase, and then the hydrogen atom was rapidly exchanged for one in the medium before its direct transfer to the substrate.     

Effects of expression of <Emphasis Type="Italic">lcb1/lcb2</Emphasis> and <Emphasis Type="Italic">lac1/lag1</Emphasis> genes on the biosynthesis of ceramides

Ceramides are important signal messenger molecules due to their role in regulating diverse responses such as cell cycle arrest, apoptosis, and senescence. Yeasts are more suitable for ceramides production than any other microorganisms since they grow fast and are non-pathogenic. However, regulations of the expression of genes involved in sphingolipid synthesis pathway are required to enhance ceramides production. In this study, we investigated the effects of co-expression of two pairs of genes that encode for serine palmitoyltransferase and ceramide synthase, respectively. Effect of other genes of the enzymes associated with the sphingolipid synthesis pathway, 3-ketosphinganine reductase and sphinganine C-4 hydroxylase were also studied and compared. The genes were cloned in to pESC-URA vector. Saccharomyces cerevisiae was cultivated aerobically in YPDG medium at 30°C. Ceramides were seperated from cell extracts by solvent extraction and quantified by HPLC with ELSD. The highest ceramides production (10.52 mg ceramides/g cell) was obtained when 3-ketosphinganine reductase, which is encoded by tsc10 gene, was overexpressed. Also, S. cerevisiae SCEL2,1 overexpressing serine palmitoyltransferase encoded by lcb2 and lcb1 genes, and S. cerevisiae SCEG1C1 overexpressing ceramide synthase encode by lag1 and lac1 genes, showed a high level of ceramides production (10.08 mg ceramides/g cell and 9.88 mg ceramides/g cell, respectively).     

Biosynthesis of acaterin: mechanism of the reaction catalyzed by dehydroacaterin reductase

Dehydroacaterin reductase is an enzyme which catalyzes the final step of acaterin biosynthesis, that is, the reduction of the C-4/C-5 double bond of dehydroacaterin. The mechanism of the reduction was investigated with a cell-free preparation obtained from the acaterin-producing microorganism, Pseudomonas sp. A 92. Incubation of dehydroacaterin in the presence of [4,4- 2H2]NADPH or D2O followed by 2H NMR analysis of the resulting acaterin revealed that the deuterium atom from NADPH was incorporated into the C-5 position of acaterin, while the deuterium atom from D2O was introduced into the C-4 position. It was further demonstrated that the pro-R hydrogen at C-4 of NADPH was stereospecifically utilized in this reduction.     

The structure and function of oestrogens. VII. The use of (9,12,12-2H3)- and (11xi,12,12-2H3) oestradiol in a test of the quinone methide hypothesis for oestrogen action

Ovariectomized mice were injected intravaginally with a physiological dose of (9,12,12-2H3)oestradiol (3), and a control group was similarly injected with (11xi,12,12-2H3)oestradiol (4). Gas-liquid chromatography/mass spectrometry (g.l.c./m.s.) analysis of the oestradiols recovered from the vaginae of the two sets of mice showed that the content and distribution of deuterium were the same as in the respective pure trideuterated oestradiols (3) and (4). This proved conclusively that the 9 alpha-hydrogen of oestradiol is not exchanged during residence in and stimulation of the vagina. It therefore appears unlikely that reversible quinone methide formation in oestradiol is the trigger mechanism for stimulation of RNA synthesis, unless a hydrogen transfer relay system permits repetitive removal and replacement of the hydrogen atom at C9 during the oxidation-reduction cycle.     

Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver.

Serine palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%.     

Stereochemistry of ornithine decarboxylase reaction

To determine the steric course of the reaction of bacterial ornithine decarboxylase [EC 4.1.1.17], we have carried out the decarboxylation of L-ornithine in 2H2O and that of DL-[2-2H]ornithine in H2O, and obtained putrescine bearing a single deuterium atom in the C-1 position. The stereochemistry of [1-2H]putrescine was established by conversion to 1-(2-pyrrolidinyl)-2-propanone with acetoacetate and the pro-S hydrogen-specific diamine oxidase from pea seedlings. Analysis of deuterium content by gas chromatography-mass spectrometry showed that the deuterium label was fully retained during the conversion of [1-2H]putrescine produced by the decarboxylation of L-ornithine in 2H2O to 1-(2-pyrrolidinyl)-2-propanone, in contrast with the considerable loss of label from [1-2H]putrescine which was produced by the decarboxylation of DL-[2-2H]ornithine in H2O. The extent of loss of the deuterium label was in good agreement with the estimated value based on the isotope effect in the diamine oxidase reaction. These results indicate that the introduced deuterium (or hydrogen) is in the pro-R position at C-1 of putrescine, and consequently the ornithine decarboxylase reaction proceeds with retention of configuration.     

Reversible cleavage of the cobalt-carbon bond to coenzyme B12 catalysed by methylmalonyl-CoA mutase from Propionibacterium shermanii. The use of coenzyme B12 stereospecifically deuterated in position 5'

1. (5'R)-(5'-2H1)Adenosine [(5'R):(5'S) = 85:15] was prepared by a procedure which involved inter alia the reduction of 6-N-benzoyl-2',3'-O-isopropylidene-5'-oxoadenosine with a reagent obtained from LiAl2H4 and (-)-isoborneol. 2. (5'S)-(5'-2H1)AdoCbl [(5'S):(5'R) = 74:26] (AdoCbl = 5'-deoxyadenosylcobalamin) was synthesized by reacting cobal(I)amin with (5'R)-2'-3'-O-isopropylidene-5'-tosyl-(5'-2H1) adenosine followed by acid hydrolysis to remove the isopropylidene protective group. 3. (5'R)-(5'-2H1)AdoCbl [(5'R):(5'S) = 77:23] was prepared by reacting cobalt(I)amin with (5'S)-5'-chloro-5'-(5'-2H1)deoxyadenosine [(5'S):(5'R) = 80:20] obtained in turn from (5'R)-(5'-2H1)adenosine. The reaction sequence involved two consecutive inversions at the C-5' atom of adenosine 4. Comparison of the 500-MHz 1H-NMR spectra of unlabelled, (5'S)- and (5'R)-(5'-2H1)AdoCbl allowed assignment of the triplet at 0.58 ppm and the doublet at 1.525 ppm to the diastereotopic 5'-HRe and 5'-HSi atoms, respectively. On acidification, these two protons gave rise to two triplets at 0.11 ppm and 1.78 ppm indicating that torsion had occurred around the C-4'--C-5' bond. 5. Samples of (5'R)- and (5'S)-(5'-2H1)AdoCbl were incubated with methylmalonyl-CoA mutase from Propionibacterium shermanii. Examination by 1H-NMR spectroscopy at 500 MHz revealed partial loss and stereochemical scrambling of the deuterium at the 5' position. This indicates transient conversion of the C-5' atom into a torsiosymmetric group and hence cleavage of the cobalt-carbon bond during interaction with the enzyme. The mechanism by which deuterium is lost remains to be elucidated.     

Sphingomyelin synthesis in Fasciola hepatica

Whole worms and/or homogenates of F. hepatica incorporate label from cytidine-5-diphospho[methyl-14C]choline,[1-14C]palmitoylCoA,[U- 14C]serine,[2-14C]methionine, [U-14C]glycine, [U-14C]threonine and [U-14C]aspartate into the various intermediates of sphingomyelin synthesis (ketosphinganine, sphinganine, sphingosine, ceramide and sphingomyelin). This suggests that sphingomyelin synthesis in F. hepatica occurs by a pathway similar to that found in mammals. However, there is some evidence that in F. hepatica 3-ketosphinganine may be N-acylated prior to reduction and dehydrogenation.     

Deuterium transfer from [1,1-2-H] ethanol during metabolism of bile acids and cyclohexanone in the isolated perfused rat liver.

Deuterium transfer from [1,1-2-H]ethanol (95 atoms % excess) to reducible substrates was studied in the isolated perfused rat liver. The dueterium excess in cyclohexanol formed from cyclohexanone was somewhat lower (49 atoms%) than found under conditions in vivo, and this was also true of the deuterium excess in lithocholic acid formed from 3-oxo-5beta-cholanoic acid. These results may reflect a slower rate of ethanol oxidation in the isolated organ than in vivo. Cycloserine decreased the dueterium transfer to both substrates, whereas addition of lactate and malate resulted in an increased deuterium excess in cyclohexanol and a decreased deuterium excess in lithocholic acid. Addition of heavy water to the perfusion fluid resulted in labelling at C-3 of lithocholic acid formed from 3-oxo-5beta-cholanoic acid, and at C-3, C-4 and C-5 of 3alpha-hydroxy-5alpha-cholanoic acid formed from 3-oxo-4-cholenoic acid. The deuterium excess of hydrogens derived from NADPH (at C-3 and C-5) was approximately the same as that of hydrogen derived directly from water (at C-4). Thus, the hydrogen of NADPH is extensively exchanged with protons of water, which explains the dilution of deuterium with protium during the transfer from [1,1-2-H]ethanol via NADPH to the bile acids. The labelling at C-5 in the reduction of the 4,5-double bond indicates that different pools of NADPH are used for reduction of this double bond and the 3-oxo group, since in a previous study it was shown that deuterium is transferred from [1,1-2-H]ethanol only in the latter reaction.     

Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes)

The activity of serine palmitoyltransferase (palmitoyl-coenzyme A [CoA]:L-serine [Ser]-C-palmitoyltransferase [decarboxylating], EC 2.3.1.50), the enzyme catalyzing the first step in the synthesis of the long-chain base required for sphingolipid assembly, has been characterized in a plant system. Enzyme activity in a microsomal membrane fraction from summer squash fruit (Cucurbita pepo L. cv Early Prolific Straightneck) was assayed by monitoring the incorporation of L-[3H]Ser into the chloroform-soluble product, 3-ketosphinganine. Addition of NADPH to the assay system resulted in the conversion of 3-ketosphinganine to sphinganine. The apparent Km for Ser was approximately 1.8 mM. The enzyme exhibited a strong preference for palmitoyl-CoA, with optimal activity at a substrate concentration of 200 [mu]M. Pyridoxal 5[prime]-phosphate was required as a coenzyme. The pH optimum was 7.6, and the temperature optimum was 36 to 40[deg]C. Enzyme activity was greatest in the microsomal fraction obtained by differential centrifugation and was localized to the endoplasmic reticulum using marker enzymes. Two known mechanism-based inhibitors of the mammalian enzyme, L-cycloserine and [beta]-chloro-L-alanine, were effective inhibitors of enzyme activity in squash microsomes. Changes in enzyme activity with size (age) of squash fruit were observed. The results from this study suggest that the properties and catalytic mechanism of Ser palmitoyltransferase from squash are similar to those of the animal, fungal, and bacterial enzyme in most respects. The specific activity of the enzyme in squash microsomes ranged from 0.57 to 0.84 nmol min-1 mg-1 of protein, values 2- to 20-fold higher than those previously reported for preparations from animal tissues.     

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.     

Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity.

We have begun a biochemical-genetic analysis of the synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae and found evidence for the occurrence of serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase, enzymes that catalyze the initial steps of the pathway in other organisms. SPT activity was demonstrated in vitro with crude membrane preparations from S. cerevisiae as judged by the formation of radiolabeled 3-ketosphinganine from the condensation of palmitoyl-coenzyme A (CoA) with radiolabeled serine. Shorter (C12 and C14) and longer (C18) acyl-CoAs sustain significant SPT activity, a result consistent with the finding of both C18 and C20 long-chain bases in the organism. Three products of the long-chain-base synthetic pathway, 3-ketosphinganine, erythrosphinganine, and phytosphingosine, neither directly inhibited the reaction in vitro nor affected the specific activity of the enzyme when these bases were included in the culture medium of wild-type cells. Thus, no evidence for either feedback inhibition or repression of enzyme synthesis could be found with these putative effectors. Mutant strains of S. cerevisiae that require a sphingolipid long-chain base for growth fall into two genetic complementation groups, LCB1 and LCB2. Membrane preparations from both lcb1 and lcb2 mutant strains exhibited negligible SPT activity when tested in vitro. Step 2 of the long-chain-base synthetic pathway was demonstrated by the stereospecific NADPH-dependent reduction of 3-ketosphinganine to erythrosphinganine. Membranes isolated from wild-type cells and from an lcb1 mutant exhibited substantial 3-ketosphinganine reductase activity. We conclude that the Lcb- phenotype of these mutants results from a missing or defective SPT, an activity controlled by both the LCB1 and LCB2 genes. These results and earlier work from this laboratory establish that SPT plays an essential role in sphingolipid synthesis in S. cerevisiae.     

Identification and characterization of 3-ketosphinganine reductase activity encoded at the BT_0972 locus in Bacteroides thetaiotaomicron

Bacterial sphingolipid synthesis is important for the fitness of gut commensal bacteria with an implied potential for regulating mammalian host physiology. Multiple steps in bacterial sphingolipid synthesis pathways have been characterized previously, with the first step of de novo sphingolipid synthesis being well conserved between bacteria and eukaryotes. In mammals, the subsequent step of de novo sphingolipid synthesis is catalyzed by 3-ketosphinganine reductase, but the protein responsible for this activity in bacteria has remained elusive. In this study, we analyzed the 3-ketosphinganine reductase activity of several candidate proteins in Bacteroides thetaiotaomicron chosen based on sequence similarity to the yeast 3-ketosphinganine reductase gene. We further developed a metabolomics-based 3-ketosphinganine reductase activity assay, which revealed that a gene at the locus BT_0972 encodes a protein capable of converting 3-ketosphinganine to sphinganine. Taken together, these results provide greater insight into pathways for bacterial sphingolipid synthesis that can aid in future efforts to understand how microbial sphingolipid synthesis modulates host-microbe interactions.     

The effect of methionine on the metabolism of serine and glycine in vitamin B-12-deficient rats.

The effect of methionine supplementation on glycine and serine metabolism was studied in vitamin B-12-deficient rats which received only 0.2% methionine in the diet. In the perfused liver, incorporation of the C-2 of glycine to the C-3 of serine was increased by addition of methionine to the perfusate. The oxidation of [1-14C]glycine to 14CO2 was however depressed. Unlike methionine, glycine did not have any significant effect on the liver folate coenzyme distribution. Oxidation of [3-14C]serine to 14CO2 both in vivo and in perfused liver was increased by methionine. A major portion of the C-3 radioactivity however was recovered in glucose. Data presented indicate that the rate of oxidation of [2-14C]histidine to 14CO2 is a more sensitive indicator of folate deficiency than the rate of oxidation of [3-14C]serine to 14CO2 although both are presumably tetrahydrofolate dependent.