Distribution of B6 vitamers in Escherichia coli as determined by enzymatic assay.

An enzymatic method for determination of B6 vitamers is presented. In this method pyridoxal 5'-phosphate is used to activate aposerine hydroxymethyltransferase to form the catalytically active holoenzyme. The active serine hydroxymethyltransferase, and two other enzymes that form a metabolic cycle, convert serine to glycine and CO2 with the concomitant production of two equivalents of NADPH. The rate of the cycle is directly proportional to the amount of active holoserine hydroxymethyltransferase, which is a measure of the amount of pyridoxal 5'-phosphate in the original sample. The cycle operates about 50 times per minute giving a 100-fold enhancement of NADPH production with respect to original pyridoxal 5'-phosphate content. Other B6 vitamers are converted to pyridoxal 5'-phosphate by a preincubation with a combination of pyridoxal kinase and pyridoxine 5'-phosphate oxidase. A complete analysis of B6 vitamers can be completed in less than 1 h and the assay is linear in the 2- to 50-pmol range of pyridoxal 5'-phosphate. The method is applied to the determination of the B6 vitamer pools in extracts of Escherichia coli. The results show that the pool of pyridoxal 5'-phosphate that is not bound to proteins is large enough to account for product inhibition of both pyridoxal kinase and pyridoxine 5'-phosphate oxidase.

The role of serine hydroxymethyltransferase isozymes in one-carbon metabolism in MCF-7 cells as determined by (13)C NMR.

The role of cytosolic and mitochondrial serine hydroxymethyltransferase in supplying one-carbon groups for purine and thymidylate biosynthesis in MCF-7 cells was investigated by observing folate-mediated one-carbon metabolism of l-[3-(13)C]serine, [2-(13)C]glycine, and [(13)C]formate. (13)C NMR was used to follow the incorporation of label into carbons 2 and 8 of purines and the methyl group attached to carbon 5 of thymidylate. The percentage enrichment of the (13)C label in purines was determined from the splitting patterns of the (1)H NMR spectra of C2 and C8 of adenine and C8 of guanine. The results show that formate is the major precursor in the cytosol of the one-carbon group in 10-formyltetrahydrofolate, which is used in purine biosynthesis, and the one-carbon group in 5,10-methylenetetrahydrofolate, which is used in thymidylate biosynthesis. Formate is formed in the mitochondria from carbon 3 of serine. The cleavage of serine to glycine and 5,10-methylenetetrahydrofolate by cytosolic serine hydroxymethyltransferase does not appear to be a major source of one-carbon groups for either purine or thymidylate biosynthesis. Carbon 3 of serine accounts for about 95% of the one-carbon pool, suggesting that other sources of one-carbon groups represent only minor pathways. [2-(13)C]Glycine is not a donor of one-carbons groups, confirming that MCF-7 cells lack a functional glycine cleavage system.

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with pyridoxal 5'-phosphate at 2.0 A resolution.

Escherichia coli pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate by the FMN oxidation of pyridoxine 5'-phosphate forming FMNH(2) and H(2)O(2). Recent studies have shown that in addition to the active site, pyridoxine 5'-phosphate oxidase contains a non-catalytic site that binds pyridoxal 5'-phosphate tightly. The crystal structure of pyridoxine 5'-phosphate oxidase from E. coli with one or two molecules of pyridoxal 5'-phosphate bound to each monomer has been determined to 2.0 A resolution. One of the pyridoxal 5'-phosphate molecules is clearly bound at the active site with the aldehyde at C4' of pyridoxal 5'-phosphate near N5 of the bound FMN. A protein conformational change has occurred that partially closes the active site. The orientation of the bound pyridoxal 5'-phosphate suggests that the enzyme catalyzes a hydride ion transfer between C4' of pyridoxal 5'-phosphate and N5 of FMN. When the crystals are soaked with excess pyridoxal 5'-phosphate an additional molecule of this cofactor is also bound about 11 A from the active site. A possible tunnel exists between the two sites so that pyridoxal 5'-phosphate formed at the active site may transfer to the non-catalytic site without passing though the solvent.

Enzymatic determination of homocysteine in cell extracts.

Determination of homocysteine levels in cells and serum is important because high homocysteine is a risk factor for cardiovascular disease. The currently used methods for homocysteine analysis either are time consuming or rely on the use of expensive equipment. Described in this study is an enzymatic assay that determines levels of homocysteine in multiple samples in less than 30 min at levels from 5 to 50 pmol using only a spectrophotometer. The reproducibility of the assay is consistent with the other methods currently used. A second assay, that is about 5-fold more sensitive, follows the enzymatic catalyzed solvent exchange of protons on glycine, which requires a scintillation counter. Both the spectrophotometric and the radiometric methods are based on the conversion of 5-methyltetrahydrofolate to tetrahydrofolate by methionine synthase. The tetrahydrofolate is formed in stoichiometric amounts to the homocysteine in the sample. In the spectrophotometric method the tetrahydrofolate is used at catalytic levels by three enzymes to form a metabolic cycle that generates NADPH from NADP(+). In the radiometric assay tetrahydrofolate is required for the enzymatic exchange of the pro 2S proton of glycine with solvent. L-Cysteine, at levels more than 30-fold higher than the upper level of homocysteine used in these assays, does not give any measurable response.

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with FMN at 1.8 A resolution.

BACKGROUND: Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate (PLP), a cofactor used by many enzymes involved in amino acid metabolism. The enzyme oxidizes either the 4'-hydroxyl group of pyridoxine 5'-phosphate (PNP) or the 4'-primary amine of pyridoxamine 5'-phosphate (PMP) to an aldehyde. PNPOx is a homodimeric enzyme with one flavin mononucleotide (FMN) molecule non-covalently bound to each subunit. A high degree of sequence homology among the 15 known members of the PNPOx family suggests that all members of this group have similar three-dimensional folds. RESULTS: The crystal structure of PNPOx from E. coli has been determined to 1.8 A resolution. The monomeric subunit folds into an eight-stranded beta sheet surrounded by five alpha-helical structures. Two monomers related by a twofold axis interact extensively along one-half of each monomer to form the dimer. There are two clefts at the dimer interface that are symmetry-related and extend from the top to the bottom of the dimer. An FMN cofactor that makes interactions with both subunits is located in each of these two clefts. CONCLUSIONS: The structure is quite similar to the recently deposited 2.7 A structure of Saccharomyces cerevisiae PNPOx and also, remarkably, shares a common structural fold with the FMN-binding protein from Desulfovibrio vulgaris and a domain of chymotrypsin. This high-resolution E. coli PNPOx structure permits predictions to be made about residues involved in substrate binding and catalysis. These predictions provide testable hypotheses, which can be answered by making site-directed mutants.

Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase.

Crystal structures of human and rabbit cytosolic serine hydroxymethyltransferase have shown that Tyr65 is likely to be a key residue in the mechanism of the enzyme. In the ternary complex of Escherichia coli serine hydroxymethyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxyl of Tyr65 is one of four enzyme side chains within hydrogen-bonding distance of the carboxylate group of the substrate glycine. To probe the role of Tyr65 it was changed by site-directed mutagenesis to Phe65. The three-dimensional structure of the Y65F site mutant was determined and shown to be isomorphous with the wild-type enzyme except for the missing Tyr hydroxyl group. The kinetic properties of this mutant enzyme in catalyzing reactions with serine, glycine, allothreonine, D- and L-alanine, and 5,10-methenyltetrahydrofolate substrates were determined. The properties of the enzyme with D- and L-alanine, glycine in the absence of tetrahydrofolate, and 5, 10-methenyltetrahydrofolate were not significantly changed. However, catalytic activity was greatly decreased for serine and allothreonine cleavage and for the solvent alpha-proton exchange of glycine in the presence of tetrahydrofolate. The decreased catalytic activity for these reactions could be explained by a greater than 2 orders of magnitude increase in affinity of Y65F mutant serine hydroxymethyltransferase for these amino acids bound as the external aldimine. These data are consistent with a role for the Tyr65 hydroxyl group in the conversion of a closed active site to an open structure.

Tight binding of pyridoxal 5'-phosphate to recombinant Escherichia coli pyridoxine 5'-phosphate oxidase.

Escherichia coli pyridoxine (pyridoxamine) 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to pyridoxal 5'-phosphate (PLP) using flavin mononucleotide (FMN) as the immediate electron acceptor and oxygen as the ultimate electron acceptor. This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli. Removal of FMN from the holoenzyme results in a catalytically inactive apoenzyme. PLP molecules bind tightly to both apo- and holoPNPOx with a stoichiometry of one PLP per monomer. The unique spectral property of apoPNPOx-bound PLP suggests a non-Schiff base linkage. HoloPNPOx with tightly bound PLP shows normal catalytic activity, suggesting that the tightly bound PLP is at a noncatalytic site. The tightly bound PLP is readily transferred to aposerine hydroxymethyltransferase in dilute phosphate buffer. However, when the PNPOx. PLP complex was added to aposerine hydroxymethyltransferase suspended in an E. coli extract the rate of reactivation of the apoenzyme was several-fold faster than when free PLP was added. This suggests that PNPOx somehow targets PLP to aposerine hydroxymethyltransferase in vivo.

Crystal structure at 2.4 A resolution of E. coli serine hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate.

Serine hydroxymethyltransferase (EC 2.1.2.1), a member of the alpha-class of pyridoxal phosphate enzymes, catalyzes the reversible interconversion of serine and glycine, changing the chemical bonding at the C(alpha)-C(beta) bond of the serine side-chain mediated by the pyridoxal phosphate cofactor. Scission of the C(alpha)-C(beta) bond of serine substrate produces a glycine product and most likely formaldehyde, which reacts without dissociation with tetrahydropteroylglutamate cofactor. Crystal structures of the human and rabbit cytosolic serine hydroxymethyltransferases (SHMT) confirmed their close similarity in tertiary and dimeric subunit structure to each other and to aspartate aminotransferase, the archetypal alpha-class pyridoxal 5'-phosphate enzyme. We describe here the structure at 2.4 A resolution of Escherichia coli serine hydroxymethyltransferase in ternary complex with glycine and 5-formyl tetrahydropteroylglutamate, refined to an R-factor value of 17.4 % and R(free) value of 19.6 %. This structure reveals the interactions of both cofactors and glycine substrate with the enzyme. Comparison with the E. coli aspartate aminotransferase structure shows the distinctions in sequence and structure which define the folate cofactor binding site in serine hydroxymethyltransferase and the differences in orientation of the amino terminal arm, the evolution of which was necessary for elaboration of the folate binding site. Comparison with the unliganded rabbit cytosolic serine hydroxymethyltransferase structure identifies changes in the conformation of the enzyme, similar to those observed in aspartate aminotransferase, that probably accompany the binding of substrate. The tetrameric quaternary structure of liganded E. coli serine hydroxymethyltransferase also differs in symmetry and relative disposition of the functional tight dimers from that of the unliganded eukaryotic enzymes. SHMT tetramers have surface charge distributions which suggest distinctions in folate binding between eukaryotic and E. coli enzymes. The structure of the E. coli ternary complex provides the basis for a thorough investigation of its mechanism through characterization and structure determination of site mutants.

Hypoglycinaemia and psychomotor delay in a child with xeroderma pigmentosum.

Glycine is a nonessential amino acid that serves as both an inhibitory and an excitatory neurotransmitter. Hyperglycinaemia occurs in non-ketotic hyperglycinaemia, a primary defect in the glycine cleavage pathway, and as a secondary feature of several inborn errors of organic acid metabolism. However, specifically low levels of glycine have never been reported. Here we report a child with complementation group C xeroderma pigmentosum (XP) characterized by a splice donor mutation in the XPC gene, multiple skin cancers and specific and persistent hypoglycinaemia. He has cognitive delay, lack of speech, autistic features, hyperactivity and hypotonia, all unexplained by the diagnosis of XP group C, a non-neurological form of the disease. Treatment with oral glycine has improved his hyperactivity. Specific hypoglycinaemia could indicate a metabolic disorder producing neurological dysfunction. Whether it is related to or coincidental with the XP is unclear.

Deamidation of asparagine residues in a recombinant serine hydroxymethyltransferase.

Serine hydroxymethyltransferase purified from rabbit liver cytosol has at least two Asn residues (Asn(5) and Asn(220)) that are 67 and 30% deamidated, respectively. Asn(5) is deamidated equally to Asp and isoAsp, while Asn(220) is deamidated only to isoAsp. To determine the effect of these Asn deamidations on enzyme activity and stability a recombinant rabbit liver cytosolic serine hydroxymethyltransferase was expressed in Escherichia coli over a 5-h period. About 90% of the recombinant enzyme could be isolated with the two Asn residues in a nondeamidated form. Compared with the enzyme isolated from liver the recombinant enzyme had a 35% increase in catalytic activity but exhibited no significant changes in either affinity for substrates or stability. Introduction of Asp residues for either Asn(5) or Asn(220) did not significantly alter activity or stability of the mutant forms. In vitro incubation of the recombinant enzyme at 37 degrees C and pH 7.3 resulted in the rapid deamidation of Asn(5) to both Asp and isoAsp with a t(1/2) of 50-70 h, which is comparable to the rate found with small flexible peptides containing the same sequence. The t(1/2) for deamidation of Asn(220) was at least 200 h. This residue may become deamidated only after some unfolding of the enzyme. The rates for deamidation of Asn(5) and Asn(220) are consistent with the structural environment of the two Asn residues in the native enzyme. There are also at least two additional deamidation events that occur during prolonged incubation of the recombinant enzyme.

Crystallization and preliminary X-ray crystallographic analysis of pyridoxine 5'-phosphate oxidase complexed with flavin mononucleotide.

Pyridoxine 5'-phosphate oxidase (PNP Ox) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate. The 53-kDa homodimeric enzyme contains a noncovalently bound flavin mononucleotide (FMN) on each monomer. Three crystal forms of Escherichia coli PNP Ox complexed with FMN have been obtained at room temperature. The first crystal form belongs to trigonal space group P3(1)21 or P3(2)21 with unit cell dimensions a = b = 64.67A, c = 125.64A, and has one molecule of the complex (PNP Ox-FMN) per asymmetric unit. These crystals grow very slowly to their maximum size in about 2 to 4 months and diffract to about 2.3 A. The second crystal form belongs to tetragonal space group P4(1) or P4(3) with unit cell dimensions a = b = 54.92A, c = 167.65A, and has two molecules of the complex per asymmetric unit. The crystals reach their maximum size in about 5 weeks and diffract to 2.8 A. A third crystal form with a rod-like morphology grows faster and slightly larger than the other two forms, but diffracts poorly and could not be characterized by X-ray analysis. The search for heavy-atom derivatives for the first two crystal forms to solve the structure is in progress.

A noncatalytic tetrahydrofolate tight binding site is on the small domain of 10-formyltetrahydrofolate dehydrogenase.

10-Formyltetrahydrofolate dehydrogenase has previously been identified as a tight binding protein of the polyglutamate forms of tetrahydrofolate (R. J. Cook and C. Wagner, Biochemistry 21, 4427-4434, 1982). Each subunit contains two independently folded domains connected by a linking peptide. By using the stable substrate and product analogs 10-formyl 5,8-dideazafolate and 5, 8-dideazafolate, respectively, we have determined that the tight binding folate site is separate from the catalytic site and that it is located on the N-terminal domain of the protein. This was achieved by cross-linking 10-formyl 5,8-dideazafolate to the dehydrogenase through the carboxyl group of the substrate analog. The cross-linked substrate analog was converted to the cross-linked product complex by adding either NADP+ or 2-mercaptoethanol, proving that the 10-formyl 5,8-dideazafolate was bound at the active site. With the active site cross-linked to 5,8-dideazafolate and not available for binding, the enzyme still bound 5, 8-dideazafolate-[3H]tetraglutamate tightly but noncovalently. Separation of the large and small domains by limited proteolysis showed that the tightly bound 5,8-dideazafolate-[3H]tetraglutamate was located on the small domain. The location of the cross-linked 10-formyl 5,8-dideazafolate at the active site was determined by amino acid sequencing of an isolated tryptic peptide.

Crystal structure of rabbit cytosolic serine hydroxymethyltransferase at 2.8 A resolution: mechanistic implications.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to form glycine and single carbon groups that are essential for many biosynthetic pathways. SHMT requires both pyridoxal phosphate (PLP) and tetrahydropteroylpolyglutamate (H4PteGlun) as cofactors, the latter as a carrier of the single carbon group. We describe here the crystal structure at 2.8 A resolution of rabbit cytosolic SHMT (rcSHMT) in two forms: one with the PLP covalently bound as an aldimine to the Nepsilon-amino group of the active site lysine and the other with the aldimine reduced to a secondary amine. The rcSHMT structure closely resembles the structure of human SHMT, confirming its similarity to the alpha-class of PLP enzymes. The structures reported here further permit identification of changes in the PLP group that accompany formation of the geminal diamine complex, the first intermediate in the reaction pathway. On the basis of the current mechanism derived from solution studies and the properties of site mutants, we are able to model the binding of both the serine substrate and the H4PteGlun cofactor. This model explains the properties of several site mutants of SHMT and offers testable hypotheses for a more detailed mechanism of this enzyme.

Crystal forms and subunit stoichiometry of serine hydroxymethyltransferase.

Serine hydroxymethyltransferase catalyzes the formation of one-carbon units essential for anabolic processes leading to the formation of such essential cellular components as purines, pyrimidines, amino acids, and lipids. Crystal structure determinations of several forms of the enzyme are under way, and to expand the comparative scope of these studies, we have crystallized the rabbit cytosolic and mitochondrial enzymes. Crystallization of serine hydroxymethyltransferase from different sources has often been problematic, and we report studies addressing these difficulties that may have more general application. The crystal lattice symmetry and the stoichiometry of the crystal asymmetric units and of the enzyme in solution suggest that serine hydroxymethyltransferase may exist as dimers, trimers, and possibly higher order complexes and that their aggregation state is affected by ionic strength.

Thermodynamic analysis of the binding of the polyglutamate chain of 5-formyltetrahydropteroylpolyglutamates to serine hydroxymethyltransferase.

The thermodynamic parameters for the binding of 5-formyltetrahydrofolate (5-CHO-H4PteGlun) and its polyglutamate forms to rabbit liver cytosolic serine hydroxymethyltransferase (SHMT) were determined by a combination of isothermal titration calorimetry and spectrophotometry. Binding of 5-CHO-H4PteGlun to SHMT exhibits both positive enthalpy and entropy, showing that binding is entropically driven. 5-CHO-H4PteGlu5 has a 300-fold increased affinity for SHMT compared to 5-CHO-H4PteGlu. This increase in affinity is due primarily to a decrease in the positive enthalpy with little change in entropy. A variety of anions inhibit the binding of 5-CHO-H4PteGlu5 with Ki values in the 10-20 mM range. Anions are ineffective inhibitors of 5-CHO-H4PteGlu binding to SHMT, showing that anions compete for the polyglutamate binding site. There was little difference in the Ki values for a series of dicarboxylic acids as inhibitors of 5-CHO-H4PteGlu5, suggesting that spacing of the negative charges may not be important in determining their effectiveness as inhibitors. Both the mono- and pentaglutamate derivatives of 5-CHO-H4PteGlun were cross-linked to SHMT by a carbodiimide reaction to Lys-450 which resides in a stretch of Lys, His, and Arg residues.

Expression, purification, and characterization of recombinant Escherichia coli pyridoxine 5'-phosphate oxidase.

A previously cloned pdxH gene from Escherichia coli coding for pyridoxine 5'-phosphate oxidase was transferred to a pET22b vector and expressed in E. coli HMS174(DE3) cells. The soluble overexpressed enzyme was rapidly purified in high yield using two chromatography columns with an overall purification of about 2.8-fold. The purified enzyme contained tightly bound FMN. The enzyme exhibited the same spectral properties and similar kinetic constants to those previously reported by G. Zhao and M. E.Winkler (J. Bacteriol. 177, 883, 1995), but differed from the properties reported by other investigators. A rapid procedure was developed for preparing apoPNP Ox in high yield. Both the holo- and apoenzymes were homodimers. The molar absorbtivity coefficient for the protein was determined for the fully active apoPNP Ox from is amino acid composition. Using this value and the spectral properties of the bound FMN it was shown by three different methods that the dimeric enzyme contains two molecules of bound FMN per dimer and not one FMN as previously reported.

Purification and characterization of recombinant rabbit cytosolic serine hydroxymethyltransferase.

A rabbit liver cDNA library in phage lambdagt10 was screened using the coding cDNA for human cytosolic serine hydroxymethyltransferase. A clone of 1754 bp was isolated and the nucleotide sequence showed an open reading frame of 1455 bp, which coded for rabbit cytosolic serine hydroxymethyltransferase and was flanked by 12 bp at the 5' end and 287 bp at the 3' end. The full-length cDNA was then cloned into a pET22b vector as a NdeI-EcoRI insert. HMS174(DE3) cells were transformed with this plasmid and, after induction with isopropyl beta-D-thiogalactopyranoside, expressed a catalytically active serine hydroxymethyltransferase. The enzyme was purified and shown to be the expressed rabbit enzyme lacking the first methionine residue. Spectral characteristics of the bound pyridoxal phosphate and kinetic constants for the natural substrates L-serine and tetrahydrofolate were essentially identical to the values obtained previously for the rabbit cytosolic enzyme. The pattern of bands shown by the pure recombinant enzyme on an isoelectric focusing gel containing 6 M urea showed a major band and a minor band representing about 15-20% of the protein. Upon incubation of the recombinant enzyme at pH 7.3 and 37 degreesC, three new bands were observed on isoelectric focusing with the concomitant formation of isoaspartyl residues, as determined by reactivity with protein isoaspartyl methyltransferase. These results are consistent with deamidation of Asn residues to isoaspartyl during the in vitro incubation. The enzyme purified from rabbit liver has previously been shown to contain isoaspartyl residues.

Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to glycine with the transfer of the one-carbon group to tetrahydrofolate to form 5,10-methylenetetrahydrofolate. No SHMT has been purified from a nonmethanogenic Archaea strain, in part because this group of organisms uses modified folates as the one-carbon acceptor. These modified folates are not readily available for use in assays for SHMT activity. This report describes the purification and characterization of SHMT from the thermophilic organism Sulfolobus solfataricus. The exchange of the alpha-proton of glycine with solvent protons in the absence of the modified folate was used as the activity assay. The purified protein catalyzes the synthesis of serine from glycine and a synthetic derivative of a fragment of the natural modified folate found in S. solfataricus. Replacement of the modified folate with tetrahydrofolate did not support serine synthesis. In addition, this SHMT also catalyzed the cleavage of both allo-threonine and beta-phenylserine in the absence of the modified folate. The cleavage of these two amino acids in the absence of tetrahydrofolate is a property of other characterized SHMTs. The enzyme contains covalently bound pyridoxal phosphate. Sequences of three peptides showed significant similarity with those of peptides of SHMTs from two methanogens.

Determination of serine hydroxymethyltransferase and reduced folate pools in tissue extracts.

Serine hydroxymethyltransferase (SHMT) from all sources tested catalyzes the slow exchange of the pro-2S proton of glycine with solvent protons. In the presence of tetrahydrofolate (H4PteGlun) this exchange rate is increased by about three orders of magnitude. This H4PteGlun-dependent exchange has been developed into a rapid and sensitive assay for both SHMT and H4PteGlun and the one-carbon derivatives of H4PteGlun. The procedure involves incubating [2-3H]glycine, H4PteGlun, and SHMT for 3 min followed by a separation of the exchanged protons in the solvent from the substrate glycine on a small Dowex-50 cation-exchange column at pH 2. In the presence of an excess of H4PteGlun the exchange rate is proportional to nanogram levels of SHMT. In the presence of an excess of SHMT the exchange rate is directly proportional to the concentration of H4PteGlun in the 0.1 to 1 pmol range. The concentration of one-carbon derivatives of H4PteGlun is determined by a preincubation of cell extracts with enzymes that convert each derivative into H4PteGlun. A complete reduced folate pool analysis of a tissue extract can be obtained in less than 2 h once a standard curve has been prepared for H4PteGlun. The method does not distinguish between mono- and polyglutamate forms of the coenzyme.