Principles of Biochemistry, Volume 2 - A. Lehninger 1985

Bioenergetics and Metabolism
Metabolism: An Overview
Metabolic pathways can be identified through direct experiments

To elucidate The sequence of Chemical Reactions that make up a given metabolic pathway, three main experimental approaches can be used. The first and most direct approach involves studying the metabolic pathway in vitro, i.e., not in living tissue itself, but in a Cell-free extract that retains The ability to catalyze the entire process under investigation. As early as the mid-nineteenth century, for example, it was known that Yeast ferment glucose into ethanol and CO₂. However, The Study of the individual steps of this metabolic pathway—which supplies anaerobic yeast Cells with almost all the energy they need—truly began only in 1898, when Eduard Buchner discovered that cell-free yeast juice obtained by pressing was also capable of fermenting glucose into ethanol and CO₂ (sec. 9.1). It was later found that Fermentation in such extracts occurs only upon The addition of inorganic phosphate and that this phosphate disappears from the extract as glucose is consumed. It turned out that some phosphorylated hexose derivative accumulates in the medium, possessing all the properties expected of an intermediate in The conversion of glucose to ethanol and CO₂. Once this intermediate was identified, an enzyme that converts it into another product was discovered in the yeast extract. This latter product was, in turn, isolated and identified. Thus, two intermediates of glucose breakdown had already been identified. By adding Enzyme Inhibitors to the extracts, researchers achieved the accumulation of other intermediates. Ultimately, through a combination of such techniques, 11 metabolites representing intermediates of Alcoholic Fermentation of glucose were isolated and identified. All 11 Enzymes catalyzing this sequence of reactions were isolated and purified. This same direct method is also used to study other metabolic pathways. It allows for the successive identification of the intermediates of a given process and the enzymes catalyzing their formation and breakdown. Once the entire sequence of reactions has been elucidated, the process can be reproduced in vitro using purified components.