Anaerobic Energy Metabolism in Yeast as a Supply-demand System

of glycolysis would lead to an increase in flux to ethanol. Tests of this prediction have been made possible by the development of recombinant-DNA technologies that allow the quantitative manipulation of the in vivo enzyme concentration profile of cells. Appropriately, one of the first of such tests was done on yeast glycolysis. Schaaff et al. (1989) over-expressed eight different enzymes of the Saccharomyces cerevisiae glycolytic and fermentative pathway by placing their genes on multicopy vectors; in so doing they were able to increase the specific enzyme activities between 3.7 and 13.9-fold. They over-expressed these enzymes singly or in pairs (phosphofructokinase/pyruvate kinase or pyruvate decarboxylase/ alcohol dehydrogenase) and measured the effect on the rate of ethanol production and the level of a number of metabolites in logarithmically growing cultures. In terms of the prevailing view that glycolytic flux-control resides in glycolysis itself the results were surprising: under no circumstances did increases in the activities of the different glycolytic enzymes significantly affect the rate of ethanol production (“and therefore the flux through glycolysis”) or affect the concentrations of “key” metabolites in comparison to the wild type. If flux-control does not reside in the “rate-limiting” enzymes, where does it reside? Schaaff and colleagues conclude that their data support the view of metabolic control analysis (Kacser et al., 1995; Heinrich and Rapoport, 1974) that the control of flux is shared among all enzymes of a metabolic system, a concept for which in general there is now ample evidence (Fell, 1996). What is not clear is what they regard as their “metabolic system”. Those trained to take the classical view would in all probability regard only the glycolytic pathway as their system and argue that even if flux-control does not reside in one or even in a few glycolytic enzymes it must still be shared among all the glycolytic enzymes, the implication being that if one simultaneously over-expressed all the enzymes of glycolysis one would theoretically expect the glycolytic flux to increase proportionally. Transport of glucose into the cell should of course also be considered part of the glycolytic apparatus; in fact, it has been suggested that substantial control on glycolytic flux is exerted by the uptake systems (Galazzo and Bailey, 1990; Bisson et al., 1993). In the remainder of this chapter I shall argue for another possibility, namely that steady-state glycolytic flux is controlled by reactions outside of what has traditionally been regarded as glycolysis, more specifically, it is controlled by those cellular processes that consume the key product of glycolysis, ATP. Furthermore, I shall argue that this is fully