A NOVEL-APPROACH FOR INVESTIGATING REACTION-MECHANISMS IN CELLS - MECHANISM OF DEOXY-TREHALOSE SYNTHESIS IN SACCHAROMYCES-CEREVISIAE STUDIED BY H-1-NMR SPECTROSCOPY

A new approach is proposed for investigating the mechanism of metaboli te synthesis in cells. This method, based on the competition between v arious substrates, allows the flux along a pathway, which is normally independent of the concentration of the corresponding precursor in the external medium, to be divided into partial fluxes. In particular, th e mechanism for the deoxy-trehalose synthesis in glucose-grown repress ed Saccharomyces cerevisiae was studied, by H-1-NMR spectroscopy, usin g the competition between 2-deoxy-D-glucose (dGlc) and 2-fluoro-deoxy- D-glucose (FdGlc) with respect to hexokinase. S. cerevisiae cells, sus pended in a standard pyrophosphate medium containing about 5x10(7) cel ls/ml, were incubated with 30 mM glucose and various concentrations of dGlc and FdGlc. Apart from dGlc6P and FdGlc6P, trehalose and the diss acharides relative to dGlc, i.e. dideoxy-trehalose (dGlc-dGlc) and deo xy trehalose (dGlc-Glc), are observed while their analogues relative t o FdGlc (FdGlc-FdGlc, FdGlc-Glc) are surprisingly absent. For the same external concentration of dGlc and FdGlc, the internal concentration of FdGlc6P is about three times larger than that of dGlc6P. The ratio of the FdGlc6P and dGlc6P concentrations is independent of the incubat ion times and proportional to the FdGlc and dGlc concentrations in the suspension. The dGlc6P concentration can thus be reduced at will by i ncreasing the [FdGlc]/[dGlc] ratio. Under these conditions, the dGlc-G lc concentration was found to vary linearly with that of dGlc6P. The p resent data clearly show that deoxy-trehalose is not synthesized from UDP-dGlc and Glc6P but from UDP-Glc and dGlc6P. This conclusion was al so confirmed by an experiment in which S. cerevisiae cells were previo usly charged with dGlc6P and then incubated with glucose.