The action of streptomycin. VI. A new metabolic intermediate.

In attempting to explain the results obtained when certain strains of Escherichia coli were treated with streptomycin, a reaction between pyruvate and oxalacetate was proposed as the sensitive reaction. The action of streptomycin on this reaction was regarded as being related to its action in inhibiting the organism since only antibiotically active forms of streptomycin were inhibitory and complete inhibition was obtained at concentrations comparable to those required to inhibit growth (Umbreit, 1949; Oginsky et al., 1949). The nature of the "oxalacetate-pyruvate" reaction has remained obscure since further studies (Umbreit et al., 1951) have shown that it was not identical with any of the known reactions of these compounds. Since the conversion of pyruvate to active acetyl and the condensation of this with oxalacetate to form citrate have been studied intensively, and since this reaction series would seem to account for the oxalacetatepyruvate effect, it was quite unlikely that a new reaction would be found. However, we were unable to demonstrate any effect of streptomycin on the known reactions of pyruvate or oxalacetate (Umbreit et al., 1951; Umbreit, 1952a,b) even at concentrations of streptomycin far above those required to inhibit growth. To the best of our knowledge no one else has been able to demonstrate inhibition of the known metabolic reactions of these compounds. One paper (Barkulis, 1951) reports streptomycin inhibition of pyruvate metabolism under anaerobic conditions, but the data available do not specify the reaction involved. Since the effect is maximal in the presence of bicarbonate, and since it may be shown that the reactions pyruvate to acetate plus formate and formate to hydrogen plus carbon dioxide are not sensitive to streptomycin per se, we have supposed that these studies pointed to a different way to measure the same reaction and were a reflection of streptomycin inhibition of the oxalacetate-pyruvate reaction. If streptomycin did not inhibit the known reactions, what was the nature of the unknown reaction? However, the experimental problem was not easily approachable because of two very large difficulties. First, it proved most difficult to obtain any kind of preparation which would contain a streptomycin sensitive reaction measurable by the rather indirect methods it was necessary to employ (either the oxidation system of Oginsky et al., 1949, or the anaerobic system of Barkulis, 1951). There were indications that this eventually would be possible since a few active preparations were obtained (Umbreit, 1949), but as an experimental tool this type of approach was, and still is, impossible. Preparations could be obtained which were capable of carrying out the known reactions of pyruvate and oxalacetate, but these reactions were not inhibited by streptomycin. It appeared that the streptomycin sensitive reaction was dependent largely upon the intact cell which would suggest either that the cell was conducting an additional reaction(s) of some quantitative significance or that the action of streptomycin had something to do with the "intactness" of the cell. Second, there was the lack of any way to measure the sensitive reaction except its inhibition by streptomycin. Under these circumstances the only way one could identify the sensitive reaction was to stop it with streptomycin so that a search for end products was fruitless. It seemed probable that one was dealing with an active intermediate, probably present in small amounts, for which one had no method of measurement except to prevent either its formation or metabolism with streptomycin. Always, however, when streptomycin showed inhibition, the presence of both pyruvate and a four-carbon dicarboxy acid was required; the latter either added or the conditions required were such as to permit its formation. Attention was turned therefore to the possibility that a seven-carbon intermediate played an important