title: Whole-cell detection by 13C NMR of metabolic flux through the C1-tetrahydrofolate synthase/serine hydroxymethyltransferase enzyme system and effect of antifolate exposure in Saccharomyces cerevisiae.
abstract: Folate-mediated one-carbon metabolism is critical for the synthesis of numerous cellular constituents required for cell growth. A potential source of one-carbon units is formate. This one-carbon unit is activated to 10-formyltetrahydrofolate via the synthetase activity of the trifunctional enzyme C1-tetrahydrofolate (THF) synthase for use in purine synthesis or can be further reduced to 5,10-methylene-THF by the dehydrogenase activity of the same enzyme. 5,10-Methylene-THF is used by serine hydroxymethyltransferase (SHMT) in the synthesis of serine. Recently, 13C NMR has been used to establish that the C1-THF synthase/SHMT enzyme system is the only route from formate to serine in vivo in the yeast Saccharomyces cerevisiae [Pasternack et al. (1992) Biochemistry 31, 8713-8719]. In vitro studies have considered the kinetics of the C1-THF synthase/SHMT enzyme system in the catalytic conversion of formate to serine [Strong et al. (1987) J. Biol. Chem. 262, 12519-12525]. In the present work, we begin to study the kinetics of this two-enzyme system in its natural environment. Provision of [13C]formate and direct detection of an intracellular accumulating pool of [3-13C]serine by 13C NMR of whole cells allow us to monitor the rate of flux through this enzyme system in vivo. The rate of accumulation of soluble [3-13C]serine under [13C]formate-saturating conditions is 13.0 +/- 1.2 microM/min relative to an external standard of serine in D2O. The extracellular formate concentration at half-maximal flux was determined to be 900 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
authors.forename: L B
authors.initials: LB
authors.lastname: Pasternack
authors.forename: D A
authors.initials: DA
authors.lastname: Laude
authors.forename: D R
authors.initials: DR
authors.lastname: Appling
mesh_terms: D000619:Aminohydrolases / Q000378:metabolism*; D002247:Carbon Isotopes; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D005574:Formate-Tetrahydrofolate Ligase / Q000378:metabolism*; D005561:Formates / Q000378:metabolism; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D012694:Serine / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D005493:Folic Acid Antagonists; D005561:Formates; D009097:Multienzyme Complexes; C030544:formic acid; D012694:Serine; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D012696:Glycine Hydroxymethyltransferase; D000619:Aminohydrolases; D005574:Formate-Tetrahydrofolate Ligase