Coupling of Reduced Pyridine Nucleotide in Leuconostoc Mesenteroides.

The demonstration that oxidation-reduction reactions mediated by the pyridine nucleotide coenzymes occur with the transfer of hydrogen between substrate and coenzyme (1) has led to the use of hydrogen isotopes to measure the occurrence of the specific coupling of reactions in the intact cell and organism (2-4). Several problems associated with the cellular organization and integration of the fermentative system of growing and nongrowing bacterial cells can be conveniently approached with this technique. It may be suggested by the common occurrence in most pathways of carbohydrate fermentation of the sequence for converting glyceraldehyde phosphate to lactic acid that these reactions may recur as an organized unit that is appended on initial sequences such as those found in the Embdem-Meyerhof, Entner-Douderoff, phosphogluconate shunt, and heterofermentative pathways. In the anaerobic breakdown of glucose by Leuconostoc mesenteroides (Fig. l), the electron balance involving glucose-6-P dehydrogenase, 6-P-gluconate dehydrogenase, and glyceraldehyde-3-P dehydrogenase in electron-donating steps, and acetate reductase, alcohol dehydrogenase, and lactate dehydrogenase in electron-accepting steps, results in the conversion of glucose to COZ (C-l), ethanol (C-2 and C-3), and lactate (C-4, C-5, and C-6). Aerobic fermentation by L. mesenteroides strain 548, which possesses an oxidase, leads to the production of COZ, acetate (instead of ethanol), and lactate but not pyruvate (5). The unabated production of lactate in the face of the thermodynamically favored aerobic oxidation of reduced pyridine nucleotide could be explained by a special relationship between lactate dehydrogenase and glyceraldehyde-3-P dehydrogenase that protects this reducing equivalent from competing reactions. L. mesenteroides is seen to be highly favored for a study with tritium-labeled glucose because the 2 C-l hydrogen atoms of ethanol and the C-2 hydrogen atom of lactate offer unique and distinguishable positions, which may be used to distinguish the hydrogen atoms transferred via reduced pyridine nucleotide generated in the oxidation of C-l of glucose by glucose-6-P dehydrogenase and C-3 by 6-P-gluconate dehydrogenase. By the