Nitric Oxide Inhibits Methionine Synthase Activity in Vivo and Disrupts Carbon Flow through the Folate Pathway

Many of nitric oxide's biological effects are mediated via NO binding to the iron in heme-containing proteins. Cobalamin (vitamin B12) is structurally similar to heme and is a cofactor for methionine synthase, a key enzyme in folate metabolism. NO inhibits methionine synthase activity in vitro, but data concerning NO binding to cobalamin are controversial. We now show spectroscopically that NO reacts with all three valency states of cobalamin and that NO's inhibition of methionine synthase activity most likely involves its reaction with monovalent cobalamin. By following incorporation of the methyl moiety of 14Cmethyltetrahydrofolic acid into protein, we show that NO inhibits methionine synthase activity in vivo, in cultured mammalian cells. The inhibition of methionine synthase activity disrupted carbon flow through the folate pathway as measured by decreased incorporation of 14Cformate into methionine, serine, and purine nucleotides. Homocysteine, but not cysteine, attenuated NO's inhibition of purine synthesis, providing further evidence that NO was acting through methionine synthase inhibition. NO's effect was observed both when NO donors were added to cells and when NO was produced physiologically in co-culture experiments. Treating cells with an NO synthase inhibitor increased formate incorporation into methionine, serine, and purines and methyl-tetrahydrofolate incorporation into protein. Thus, physiological concentrations of NO appear to regulate carbon flow through the folate pathway. Many of nitric oxide's biological effects are mediated via NO binding to the iron in heme-containing proteins. Cobalamin (vitamin B12) is structurally similar to heme and is a cofactor for methionine synthase, a key enzyme in folate metabolism. NO inhibits methionine synthase activity in vitro, but data concerning NO binding to cobalamin are controversial. We now show spectroscopically that NO reacts with all three valency states of cobalamin and that NO's inhibition of methionine synthase activity most likely involves its reaction with monovalent cobalamin. By following incorporation of the methyl moiety of 14Cmethyltetrahydrofolic acid into protein, we show that NO inhibits methionine synthase activity in vivo, in cultured mammalian cells. The inhibition of methionine synthase activity disrupted carbon flow through the folate pathway as measured by decreased incorporation of 14Cformate into methionine, serine, and purine nucleotides. Homocysteine, but not cysteine, attenuated NO's inhibition of purine synthesis, providing further evidence that NO was acting through methionine synthase inhibition. NO's effect was observed both when NO donors were added to cells and when NO was produced physiologically in co-culture experiments. Treating cells with an NO synthase inhibitor increased formate incorporation into methionine, serine, and purines and methyl-tetrahydrofolate incorporation into protein. Thus, physiological concentrations of NO appear to regulate carbon flow through the folate pathway. cbl(II), or cbl(III), cobalamin in the +1, +2, or +3 valency state, respectively S-adenosylmethionine baby hamster kidney cells bovine serum albumin Dulbecco's modified Eagle's medium S-nitrosoglutathione N G-nitro-l-arginine-methyl ester nitric oxide nitrous oxide S-nitroso-N-acetylpenicillamine propylamine propylamine NONOate Vitamin B12 deficiency leads to pernicious anemia and subacute combined degeneration of the spinal cord (1Babior B.M. Beutler E. Lichtman M.A. Coller B.S. Lipps T.J. Williams Hematology. McGraw-Hill Inc., New York1995Google Scholar). Pernicious anemia is characterized by megaloblastic erythropoiesis and is secondary to decreased activity of methionine synthase, one of two mammalian enzymes that requires vitamin B12 (cobalamin) as a cofactor (2Banerjee R.V. Matthews R.G. FASEB J. 1990; 4: 1450-1459Crossref PubMed Scopus (282) Google Scholar, 3Ludwig M.L. Matthews R.G. Annu. Rev. Biochem. 1997; 66: 269-313Crossref PubMed Scopus (293) Google Scholar). Methionine synthase catalyzes the transfer of the methyl group of 5-methyltetrahydrofolate to homocysteine via a methylcobalamin intermediate with cycling of cobalamin between the +1 valency state (i.e.cbl(I))1 and the +3 valency state (i.e. cbl(III)) (2Banerjee R.V. Matthews R.G. FASEB J. 1990; 4: 1450-1459Crossref PubMed Scopus (282) Google Scholar, 3Ludwig M.L. Matthews R.G. Annu. Rev. Biochem. 1997; 66: 269-313Crossref PubMed Scopus (293) Google Scholar). Methyltetrahydrofolate is the major intracellular storage form of folates, and its synthesis from 5,10-methylene tetrahydrofolate is essentially irreversible in vivo (2Banerjee R.V. Matthews R.G. FASEB J. 1990; 4: 1450-1459Crossref PubMed Scopus (282) Google Scholar, 4Rosenblatt D.S. Cooper B.A. Lue-Shing S. Wong P.W.K. Berlow S. Narisawa K. Baumgartner R. J. Clin. Invest. 1979; 63: 1019-1025Crossref PubMed Scopus (36) Google Scholar) (Fig. 1). Thus, decreased methionine synthase activity leads to trapping of intracellular folates as 5-methyltetrahydrofolate, and the megaloblastic anemia of vitamin B12 deficiency is virtually indistinguishable from the megaloblastosis of folate deficiency (1Babior B.M. Beutler E. Lichtman M.A. Coller B.S. Lipps T.J. Williams Hematology. McGraw-Hill Inc., New York1995Google Scholar). Nitric oxide (NO) is produced by most cell types and regulates a diverse array of biological functions (5Ignarro L. Murad F. Nitric Oxide: Biochemistry, Molecular Biology, and Therapeutic Implications. Academic Press, Inc., San Diego1995Google Scholar, 6Lloyd-Jones D.M. Bloch K.D. Annu. Rev. Med. 1996; 47: 365-375Crossref PubMed Scopus (215) Google Scholar). NO has been reported to inhibit methionine synthase activity in vitro (7Brouwer M. Chamulitrat W. Ferruzzi G. Sauls D.L. Weinberg J.B. Blood. 1996; 88: 1857-1864Crossref PubMed Google Scholar, 8Nicolaou A. Warefield C.J. Kenyon S.H. Gibbons W.A. Eur. J. Biochem. 1997; 244: 876-882Crossref PubMed Scopus (30) Google Scholar, 9Nicolaou A. Kenyon S.H. Gibbons J.M. Ast T. Gibbons W.A. Eur. J. Clin. Invest. 1996; 26: 167-170Crossref PubMed Scopus (61) Google Scholar), and it might be expected to bind to the cobalt in cobalamin because (i) NO binds tightly to the iron in heme (10Kharitonov V.G. Bonaventura J. Sharma V.S. Feelisch M. Stamler J.S. Methods in Nitric Oxide Research. John Wiley (ii) ferrous heme and cbl(III) are isoelectronic; and (iii) in both heme and cobalamin, the metal ion is coordinated to four in-plane nitrogen atoms of a tetrapyrrole ring and has two out-of-plane ligands (3Ludwig M.L. Matthews R.G. Annu. Rev. Biochem. 1997; 66: 269-313Crossref PubMed Scopus (293) Google Scholar). However, conflicting results have been published concerning NO binding to cobalamin: Bauer (11Bauer J.A. Anti-Cancer Drugs. 1998; 9: 239-244Crossref PubMed Scopus (31) Google Scholar) reported that NO binds to divalent cobalamin (i.e. cbl(II)); Brouweret al. (7Brouwer M. Chamulitrat W. Ferruzzi G. Sauls D.L. Weinberg J.B. Blood. 1996; 88: 1857-1864Crossref PubMed Google Scholar) found that NO binds to both cbl(II) and (III); Firth et al. (12Firth R.A. Hill H.A.O. Pratt J.M. Thorp R.G. Williams R.J.P. J. Chem. Soc. 1969; A: 381-386Crossref Google Scholar) found no evidence of NO binding to either cbl(II) or (III); and Rochelle and associates (13Rochelle L.G. Morana S.J. Kruszyna H. Russell M.A. Wilcox D.E. Smith R.P. J. Pharmacol. Exp. Ther. 1995; 275: 48-52PubMed Google Scholar, 14Kruszyna H. Magyar J.S. Rochelle L.G. Russell M.A. Smith R.P. Wilcox D.E. J. Pharmacol. Exp. Ther. 1998; 285: 665-671PubMed Google Scholar) initially reported that NO binds to cbl(III) and oxidizes cbl(II) to cbl(III) but subsequently concluded that NO does not react with either species. There have been no studies of NO reaction with cbl(I), but nitrous oxide (N2O), another oxide of nitrogen, inhibits methionine synthase activity by oxidizing cbl(I) to cbl(II), and inhalation of this gas is the most common cause of acute megaloblastosis (1Babior B.M. Beutler E. Lichtman M.A. Coller B.S. Lipps T.J. Williams Hematology. McGraw-Hill Inc., New York1995Google Scholar, 15Drummond J.T. Matthews R.G. Biochemistry. 1994; 33: 3732-3741Crossref PubMed Scopus (79) Google Scholar). We found that NO reacts with all three oxidation states of cobalamin but that its mechanism of methionine synthase inhibition appears to be similar to that of N2O, i.e. oxidation of cbl(I) to cbl(II). We show that NO inhibits methionine synthase activityin vivo and that NO produced by three different pharmacological agents or produced physiologically by rat C6 glioma cells inhibits carbon flow through the folate pathway. Contrarily, an NO synthase inhibitor increased carbon flow through folates. Thus, the data suggest that NO may modulate folate-mediated one-carbon transfer reactions. Cbl(III)-OH (Sigma; acetate salt) was prepared in 0.1m sodium phosphate, pH 7.0, in a spectrophotometer cell sealed with a rubber septum and deoxygenated by passing argon through the solution for 30 min. Cbl(II) and cbl(I) were produced from deoxygenated cbl(III) solutions using 300 µmdithiothreitol and zinc dust suspended in 10% aqueous ammonium chloride, respectively (16Schrauzer G.N. Holland R.J. J. Am. Chem. Soc. 1971; 93: 4060-4062Crossref PubMed Scopus (63) Google Scholar). Chemically pure grade NO gas (minimum 99% purity; Matheson Gas Products) was passed through 1 m NaOH immediately prior to use to remove contaminating nitrogen oxide species; it was then added anerobically to cobalamin solutions. Similar results were obtained when NO was generated from the NO donor PAPA-NONOate (Cayman Chemical). Absorption spectra were obtained using a dual beam Kontron 860 spectrophotometer. CS-54 rat pulmonary artery smooth muscle cells, which maintain differentiated properties through multiple subcultures, were obtained from A. Rothman (University of California, San Diego) (17Rothman A. Kulik T.J. Taubman M.B. Berk B.C. Smith W.J. Nadal-Ginard B. Circulation. 1992; 86: 1977-1986Crossref PubMed Google Scholar). HL-60 human promyelocytic leukemic cells and baby hamster kidney (BHK) fibroblasts were obtained from the American Tissue Culture Collection, and C6 rat glioma cells were obtained from M. Ellisman (University of California, San Diego). All four cell lines were routinely cultured as described previously: CS-54, BHK, and C6 cells in Dulbecco's modified Eagle's medium (DMEM) and HL-60 cells in RPMI 1640 medium (18Scheele J.S. Pilz R.B. von Lintig F.C. Boss G.R. Oncogene. 1998; 17: 2211-2223Crossref PubMed Scopus (7) Google Scholar, 19Qiu W. Zhuang S. von Lintig F.C. Boss G.R. Pilz R.B. J. Biol. Chem. 2000; 275: 31921-31929Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 20Idriss S.D. Gudi T. Casteel D.E. Kharitonov V.G. Pilz R.B. Boss G.R. J. Biol. Chem. 1999; 274: 9489-9493Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Both media were supplemented with 10% bovine which as a of cobalamin. were using supplemented with and bovine serum albumin which be to as was because it incorporation of into methionine of protein. acid but purines and and cells not in it because of of serum were in the medium for CS-54 cells were from medium into the medium and were for 30 in the or of or N G-nitro-l-arginine-methyl ester The cells were then for with of acid medium the of the cells were for in the were to for 30 and for 30 and of and was as described G.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the was by and the was with from 1 to and with cell from to of methionine and synthesis were measured as described following 14Cformate incorporation into methionine and in G.R. J. Clin. Invest. PubMed Scopus Google Scholar, G.R. J. Clin. Invest. PubMed Scopus Google Scholar, G.R. Pilz R.B. J. Clin. Invest. PubMed Scopus Google Scholar) HL-60 cells a of 1 or or CS-54 cells were to the for the in cells were to and The cells were for 30 in the or of S-nitroso-N-acetylpenicillamine S-nitrosoglutathione or were then for with of 14Cformate medium The cells were as described for incorporation into protein, but and the were the of and was three in 10% in and for The was and the was in Methionine was from by in a and were with by The were with from 1 to and with cell from 1 to be with the cells were in medium of purine as described similar results were obtained when the medium the in homocysteine was from the were for 30 in the or of as described for of methionine and synthesis, and then of and purine synthesis were measured as described G.R. J. Biol. Chem. Full Text PDF PubMed Google R.B. Boss G.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, G.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). for purine synthesis, cells were with of 14Cformate for 1 and then in the cell were for and the purine were by ion into the was by and the data are as cells. The was with from to and with cell from 1 to purine synthesis by the cells were with of medium for 1 and then three in The cells were in and in the was measured by The was with and cell and we have that this the incorporation of into with of the as intracellular R.B. Boss G.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, G.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). that were either or rat C6 glioma cells that been to were added 1 HL-60 cells in 1 of of the was added an NO and a the cells were with 14Cformate as described of methionine, serine, and purine synthesis were measured in HL-60 cells by the cells with three with we by cell that of the HL-60 cells were and by that the of C6 cells was described for cell types R.P. R.J. Biochem. J. PubMed Scopus Google Scholar), we found in that C6 glioma cells a of purines into the medium and that the of purines was of the of purines by the HL-60 cells the Thus, it is that the C6 cells to the of purine synthesis by HL-60 cells. NO binds to cobalamin, we studies of NO with all three oxidation states of cobalamin. We found that NO a but in the of (Fig. similar to that found by et al. (7Brouwer M. Chamulitrat W. Ferruzzi G. Sauls D.L. Weinberg J.B. Blood. 1996; 88: 1857-1864Crossref PubMed Google Scholar). NO to cbl(III) were to NO binding to We NO to this by that cbl(III) was by a that of cbl(III) a been with et M. Chamulitrat W. Ferruzzi G. Sauls D.L. Weinberg J.B. Blood. 1996; 88: 1857-1864Crossref PubMed Google Scholar), we found no effect of NO the spectra of or not NO binds to cbl(II) and cbl(I), we generated two from using to cbl(II) and zinc dust suspended in ammonium to cbl(I) (16Schrauzer G.N. Holland R.J. J. Am. Chem. Soc. 1971; 93: 4060-4062Crossref PubMed Scopus (63) Google Scholar). NO to cbl(II) its of (Fig. passing argon through this solution the of cbl(II). NO to cbl(I) (Fig. immediately the to that of a the of Thus, NO binds to both cbl(II) and cbl(III), and it oxidizes cbl(I) to cbl(II). the following three concerning NO's with i.e. of binding to and binding to cbl(II), and reaction with cbl(I) suggest a mechanism NO inhibits methionine synthase of methionine synthase activity in cell and in enzyme by NO concentrations (7Brouwer M. Chamulitrat W. Ferruzzi G. Sauls D.L. Weinberg J.B. Blood. 1996; 88: 1857-1864Crossref PubMed Google Scholar, 8Nicolaou A. Warefield C.J. Kenyon S.H. Gibbons W.A. Eur. J. Biochem. 1997; 244: 876-882Crossref PubMed Scopus (30) Google Scholar, 9Nicolaou A. Kenyon S.H. Gibbons J.M. Ast T. Gibbons W.A. Eur. J. Clin. Invest. 1996; 26: 167-170Crossref PubMed Scopus (61) Google Scholar) does not physiological concentrations of NO inhibit the enzyme in We methionine synthase activity in vivo by incorporation into in CS-54 pulmonary smooth muscle cells. this between incorporation of into via methionine as an acid or via it is methionine synthase activity (Fig. 1). this we found that PAPA-NONOate decreased incorporation of into protein, from in the of the to cells in the of the S.D. of three in Similar results were found in C6 glioma cells. Thus, NO inhibits methionine synthase activity in methionine synthase activity not methionine synthesis from but because of trapping of folates as 5-methyltetrahydrofolate, it carbon flow through the folate pathway and synthesis (2Banerjee R.V. Matthews R.G. FASEB J. 1990; 4: 1450-1459Crossref PubMed Scopus (282) Google Scholar, G.R. J. Clin. Invest. PubMed Scopus Google Scholar) (Fig. 1). this we measured 14Cformate incorporation into methionine and in HL-60 human leukemic cells and as described this in vivo of methionine and synthesis and carbon through the folate pathway G.R. J. Clin. Invest. PubMed Scopus Google G.R. J. Clin. Invest. PubMed Scopus Google Scholar). We found that three different NO and concentrations to have physiological effects T. K. T. K. K. J. 1997; PubMed Scopus Google Scholar, T. M. Gibbons 1996; PubMed Scopus Google Scholar), decreased methionine and synthesis in both cell types a and the effect of is not but was similar to the two NO Methionine synthesis was decreased by in HL-60 cells and by in cells, and synthesis was decreased by in both HL-60 and cells (Fig. a of and effect methionine or The inhibition of methionine and synthesis by the three NO donors was not to of the or to inhibition of synthesis, because the of the cell as measured by of synthesis as measured by incorporation into were folates as purine synthesis by the two in the pathway (Fig. 1). We found that the three NO donors of purine synthesis by in HL-60 cells and in cells (Fig. the data for and PAPA-NONOate are HL-60 cells, PAPA-NONOate purine synthesis a as as and inhibition not The inhibition purine synthesis by PAPA-NONOate was when the was from cells prior to of purine synthesis observed in cells (Fig. were in medium supplemented with similar results were obtained when the cells were in medium physiological concentrations (i.e. of was observed in the methionine and synthesis decreased of purine synthesis in HL-60 or cells. The three NO donors no effect purine synthesis by the pathway as measured by incorporation into similar data were obtained for HL-60 The data that NO not the of the major for purine synthesis, intracellular is tightly G.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, G.R. Pilz R.B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). NO decreased of synthesis by methionine synthase, then purine synthesis in the of the of methionine synthase, the of inhibition by NO We in CS-54 cells and in the of an purine synthesis when homocysteine was from the medium (Fig. with data are with that for an acid purine synthesis G.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and that homocysteine in PAPA-NONOate purine synthesis both in cells cultured in the or of However, the two were to of and PAPA-NONOate in the and of cysteine, or to the medium was effect are for Thus, homocysteine the effect of NO as an inhibitor of purine synthesis, by providing and the of the methionine synthase the in and homocysteine was in the the observed effects were not secondary to a deficiency of physiological of NO produced by cells have a similar effect as the NO donors methionine, serine, purine synthesis, we co-culture using rat C6 glioma cells as a of cells as a have NO synthase, and NO D.L. E. Nitric 1997; PubMed Scopus Google Scholar). We by and concentrations in the medium S.D. Gudi T. Casteel D.E. Kharitonov V.G. Pilz R.B. Boss G.R. J. Biol. Chem. 1999; 274: 9489-9493Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), that C6 cells produced NO a of cells, which with results from studies D.L. E. Nitric 1997; PubMed Scopus Google Scholar). of HL-60 cells with C6 cells decreased of 14Cformate incorporation into methionine, serine, and purine in HL-60 cells by and added to the medium as an NO the effects of C6 cells methionine, serine, and purine synthesis in HL-60 cells no effect HL-60 cells cultured in the of C6 cells (Fig. a and of NO by the NO donors are not in the in the effect of the with the co-culture is with NO the the C6 cells. further the effect of physiologically produced NO methionine, serine, and purine synthesis, we CS-54 cells with a NO synthase were in and a of increased methionine, serine, and purine synthesis with we found that increased incorporation into protein, from to cells S.D. of three in NO a in different physiological and (5Ignarro L. Murad F. Nitric Oxide: Biochemistry, Molecular Biology, and Therapeutic Implications. Academic Press, Inc., San Diego1995Google Scholar, 6Lloyd-Jones D.M. Bloch K.D. Annu. Rev. Med. 1996; 47: 365-375Crossref PubMed Scopus (215) Google Scholar). Many of NO's effects and are mediated via NO binding to the iron in the heme group of which the enzyme and the intracellular of the J.S. 1994; Full Text PDF PubMed Scopus Google Scholar, M.A. Russell R.J. S. S. S. A. 1996; 93: PubMed Scopus Google Scholar). NO has a for ferrous heme with a binding the of and NO binds to heme Sharma V.S. Biochemistry. 1992; PubMed Scopus Google Scholar). and cobalt are in the and the ring of heme and the ring of cobalamin are both tetrapyrrole Hematology. Williams A: 381-386Crossref Google Scholar, 14Kruszyna H. Magyar J.S. Rochelle L.G. Russell M.A. Smith R.P. Wilcox D.E. J. Pharmacol. Exp. Ther. 1998; 285: 665-671PubMed Google Scholar), are and NO we for the that NO reacts and with and studies NO to cbl(II) and cbl(III) be published We the of following 14Cformate incorporation into methionine, serine, and purine G.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, G.R. J. Clin. Invest. PubMed Scopus Google Scholar, G.R. J. Clin. Invest. PubMed Scopus Google Scholar, G.R. Pilz R.B. J. Clin. Invest. PubMed Scopus Google Scholar, R.B. Boss G.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, G.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. J. M. J. 1990; PubMed Scopus Google Scholar) to show that NO carbon flow through the folate pathway in different types of human and cells, smooth muscle cells. NO's inhibition of carbon flow through folate was secondary to its methionine synthase activity because (i) have that NO is a inhibitor of methionine synthase activity in vitro (7Brouwer M. Chamulitrat W. Ferruzzi G. Sauls D.L. Weinberg J.B. Blood. 1996; 88: 1857-1864Crossref PubMed Google Scholar, 8Nicolaou A. Warefield C.J. Kenyon S.H. Gibbons W.A. Eur. J. Biochem. 1997; 244: 876-882Crossref PubMed Scopus (30) Google Scholar, 9Nicolaou A. Kenyon S.H. Gibbons J.M. Ast T. Gibbons W.A. Eur. J. Clin. Invest. 1996; 26: 167-170Crossref PubMed Scopus (61) Google Scholar); (ii) we found that NO donors decreased incorporation into and (iii) we that homocysteine the of PAPA-NONOate as an inhibitor of We the that purine synthesis by is to inhibit M. J. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar), which synthesis and cause purine to the inhibit purine synthesis by a the concentrations the NO donors not appear to because no effect incorporation into or incorporation into purine nucleotides. There are enzymes that cobalamin as a in and in mammalian two methionine synthase and are to cobalamin (3Ludwig M.L. Matthews R.G. Annu. Rev. Biochem. 1997; 66: 269-313Crossref PubMed Scopus (293) Google Scholar). to the of tetrahydrofolate from 5-methyltetrahydrofolate, methionine synthase is one of four enzymes in the cycling of methyl between and homocysteine (2Banerjee R.V. Matthews R.G. FASEB J. 1990; 4: 1450-1459Crossref PubMed Scopus (282) Google Scholar) (Fig. 1). is the of methyl for a of biological that and M. E. G. J. Biol. Chem. Full Text PDF PubMed Google Scholar); in propylamine for synthesis M. E. G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the reaction by methionine synthase, the methyl group of 5-methyltetrahydrofolate is to cobalamin, a intermediate (3Ludwig M.L. Matthews R.G. Annu. Rev. Biochem. 1997; 66: 269-313Crossref PubMed Scopus (293) Google Scholar). of the cbl(I), because of its oxidation state, be to cbl(II) (3Ludwig M.L. Matthews R.G. Annu. Rev. Biochem. 1997; 66: 269-313Crossref PubMed Scopus (293) Google Scholar). human methionine synthase has been that to from cbl(II) A. R. J.M. D.S. R.A. S. A. 1998; PubMed Scopus Google Scholar). Thus, the methionine synthase reaction is and because of its to it is likely to be data suggest that NO may as a physiological of methionine synthase, because we found that produced NO methionine, serine, and purine synthesis and that an NO synthase inhibitor increased incorporation into and increased 14Cformate incorporation into methionine, serine, and purine nucleotides. There are two NO's with cobalamin inhibit methionine synthase activity and with carbon flow through the folate pathway. NO bind to the methyl binding of cbl(III), or of the intermediate in the methionine synthase NO cbl(I) to cbl(II) and with the cbl(I) cbl(III) that is an of the methionine synthase We found no evidence that NO with the methyl group in and the binding of cbl(II) with or is J. PubMed Scopus Google Scholar) as to binding of NO to cbl(II). Thus, it appears that of NO with cbl(III) or cbl(II) or no in enzyme inhibition. NO inhibits methionine synthase by with cbl(I), then data that NO have or no effect the which involves cbl(II) and cbl(III) oxidation N2O, appears to inhibit methionine synthase activity by with cbl(I), the of enzyme inhibition may be different between the two the by with of cobalamin a secondary NO of the NO is a and inhibitor of NO is to be D.M. Bloch K.D. Annu. Rev. Med. 1996; 47: 365-375Crossref PubMed Scopus (215) Google Scholar, S. A. New J. Med. PubMed Scopus Google Scholar). The inhibition of methionine synthesis we observed that NO the intracellular the of and because of NO's effect the may be G.N. J. J. Med. 1998; PubMed Scopus Google Scholar). it be to NO leads to