Glycolysis in Cell-free Systems

Eduard Buchner’s discovery of cell-free metabolism 100 years ago was a landmark in biochemistry; it can be said to be the start of detailed metabolic studies. The fact that a soluble fraction of yeast, which he called “zymase”, was able to produce ethanol from glucose was revolutionary, and finally put paid to the idea that living cells were essential for fermentation (Buchner, 1897). Later, this proteincontaining extract was recognized as being a complex mixture containing enzymes responsible for the process we now call glycolysis, and over the next half-century all the metabolites and enzymes of glycolysis were described. This classic biochemistry of using a cellfree extract to catalyse a biological process has formed the basis of experiments that have occupied many of my research efforts over the past 30 years. One feature of Buchner’s experiments was that cell-free systems never produced anything like the conversion of sugars to alcohol that whole yeast cells can achieve. Even his very concentrated extract, equivalent to a thick slurry of yeast cells, produced carbon dioxide and alcohol at a rather slow rate, which we can retrospectively estimate as less than 1% of the potential enzyme activity. Typically in the production of wine, the starting liquor contains about 20% by weight of sugars, which are converted completely to ethanol, CO2, yeast biomass and the all-important minor flavour components. But with the early experiments with yeast extracts, only a small amount of the added sugar could be converted to ethanol before the process stopped after a day or two. One of the principal reasons is that the control of glucose entry to the glycolytic pathway is, at least in part, provided by the yeast cell membrane, which of course is lost in making the cellfree extracts. We can now appreciate that the restrained metabolism was due mainly to uncontrolled phosphorylation of glucose, and the