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

Idrees O Danishpajooh,Tanima Gudi,Yongchang Chen,Vladimir G Kharitonov,Vijay S Sharma,Gerry R Boss

doi:10.1074/jbc.m104043200

Idrees O Danishpajooh, Tanima Gudi + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m104043200

Copy DOI

Abstract

Many of nitric oxide's biological effects are mediated via NO binding to the iron in heme-containing proteins. Cobalamin (vitamin B(12)) 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 [(14)C]methyltetrahydrofolic 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 [(14)C]formate 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.

Highlights

Vitamin B12 deficiency leads to pernicious anemia and subacute combined degeneration of the spinal cord [1]
We show that Nitric oxide (NO) inhibits methionine synthase activity in 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
We found that NO caused a small but definite change in the spectrum of cbl(III)-OH (Fig. 2a), similar to that found by Brouwer et al [7]

Summary

EXPERIMENTAL PROCEDURES

Generation of Cobalamin Derivatives and Absorption Spectra— Cbl(III)-OH (Sigma; acetate salt) was prepared in 0.1 M 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. Experiments were performed using methionine-free DMEM supplemented with 200 ␮M DL-homocysteine thiolactone (Sigma) and 0.2% bovine serum albumin (BSA) This medium, which will be referred to as experimental medium, was used because it allowed optimal incorporation of radioactive label into methionine residues of protein. The cells were shaken gently at 37 °C for 30 min in the absence or presence of 100 ␮M PAPA-NONOate, 200 ␮M S-nitroso-Nacetylpenicillamine (SNAP; Alexis Biochemicals), 100 ␮M S-nitrosoglutathione (GS-NO; Calbiochem), or 2 mM NAME They were incubated for 2 h with 20 ␮Ci of [14C]formate (Moravek Radiochemicals; 52 mCi/mmol, final medium concentration 190 ␮M). Measurement of Rates of de Novo and Salvage Purine Nucleotide Synthesis—To be strictly comparable with the above experiments, cells were incubated in experimental medium during measurement of purine nucleotide synthesis; as described later, similar results were obtained when the medium contained methionine. It is unlikely that the C6 cells contributed significantly to the measurement of purine synthesis by HL-60 cells

RESULTS

NO Disrupts Carbon Flow through Folate Intermediates

DISCUSSION