METABOLIC RESPONSES OF PYRUVATE DECARBOXYLASE-NEGATIVE SACCHAROMYCES-CEREVISIAE TO GLUCOSE EXCESS

Saccharomyces cerevisiae, oxidation of pyruvate to acetyl coenzyme A c an occur via two routes. In pyruvate decarboxylase-negative (Pdc(-)) m utants, the pyruvate dehydrogenase complex is the sole functional link between glycolysis and the tricarboxylic acid (TCA) cycle. Such mutan ts therefore provide a useful experimental system with which to study regulation of the pyruvate dehydrogenase complex. In this study, a pos sible in vivo inactivation of the pyruvate dehydrogenase complex was i nvestigated. When respiring, carbon-limited chemostat cultures of wild -type S. cerevisiae were pulsed with excess glucose, an immediate onse t of respiro-fermentative metabolism occurred, accompanied by a strong increase of the glycolytic flux. When the same experiment was perform ed with an isogenic Pdc(-) mutant, only a small increase of the glycol ytic flux was observed and pyruvate was the only major metabolite excr eted. This finding supports the hypothesis that reoxidation of cytosol ic NADH via pyruvate decarboxylase and alcohol dehydrogenase is a prer equisite for high glycolytic fluxes in S. cerevisiae. In Pdc(-) cultur es, the specific rate of oxygen consumption increased by ca. 40% after a glucose pulse. Calculations showed that pyruvate excretion by the m utant was not due to a decrease of the pyruvate flux into the TCA cycl e. We therefore conclude that rapid inactivation of the pyruvate dehyd rogenase complex (e.g., by phosphorylation of its E1 alpha subunit, a mechanism demonstrated in many higher organisms) is not a relevant mec hanism in the response of respiring S. cerevisiae cells to excess gluc ose. Consistently, pyruvate dehydrogenase activities in cell extracts did not exhibit a strong decrease after a glucose pulse.