WHOLE-CELL DETECTION BY C-13 NMR OF METABOLIC FLUX THROUGH THE C-1-TETRAHYDROFOLATE SYNTHASE/SERINE HYDROXYMETHYLTRANSFERASE ENZYME-SYSTEM AND EFFECT OF ANTIFOLATE EXPOSURE IN SACCHAROMYCES-CEREVISIAE

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 C-1-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, C-13 NMR has been used to establish that the C-1-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 C-1-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 enviroment. Provision of [C-13]formate and direct detection of an intracellular accumulating pool of [3-C-13]serine by C-13 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-C-13] serine under [C-13] formate-saturating conditions is 13.0 +/- 1.2 mu M/min relative to an external standard of serine in D2O. The extracellular formate concentration at half-maximal flux was determined to be 900 mu M. Direct observation of the effect of THF depletion induced by exposure to methotrexate and sulfonilamide indicates folate cofactor availability to be the rate-limiting factor in this enzyme system in vivo under formate-saturating conditions. The profile of loss of activity of the C-1-THF synthase/SHMT enzyme system reveals a compartmentation of reduced folate pools available to thymidylate synthase and the C-1-THF synthase/SHMT enzyme system. Modeling of non-steady-state kinetics through C-1-THF synthase/SHMT predicts the effective local THF pool concentration to be less than 1.0 mu M at C-1-THF synthase/SHMT in vivo.