The metabolism of Prototheca zopfii was investigated in an attempt to establish the specific function of its growth factor, thiamin. A study of the oxidative decomposition of various substrates by this organism demonstrated that the addition of catalytic amounts of thiamin to vitamin-deficient cells causes a pronounced stimulation in the rate of oxygen utilization during the degradation of certain compounds.
The phosphoric ester of thiamin is known to be the prosthetic group of carboxylase. The fact that this enzyme is involved in the decomposition of pyruvic acid suggested that this α-keto acid might be an important intermediate product in the metabolism of Prototheca. Pyruvic acid, however, was not included in the list of organic substances which Barker had reported as utilized by this alga. Barker's observations were confirmed, but subsequent experiments led to serious doubts as to the validity of his interpretation. Further investigations resulted in the establishment of environmental conditions which permit this alga to readily decompose pyruvic acid, as well as nearly all other organic acids tested. This can be accomplished by providing a millieu of sufficiently low pH to insure the presence of undissociated acid molecules.
The stimulatory effect on the rate of oxygen consumption, caused by the addition of minute amounts of thiamin to suspensions of vitamin-deficient cells of Prototheca respiring pyruvic acid, indicates that the presence of thiamin results in the synthesis of enzyme systems which are involved in the decomposition of pyruvic acid.
Experimental data on the oxidation of pyruvic acid and other organic compounds are discussed in the light of various hypotheses which have been advanced concerning the rôle of carboxylase in the decomposition of pyruvic acid. The conservative conclusion which can be drawn from the available information is that there appears to be no justification for a belief that thiamin and carboxylase are functional in biochemical reactions other than in decarboxylation and carboxylation processes.
The discovery of the ability of Prototheca to utilize substituted and dicarboxylic acids led to further studies on the mechanism of oxidative assimilation. The results of these investigations are in agreement with those of Clifton and Logan, and of Doudoroff, and indicate the existence of a relatively simple chemical mechanism of assimilation rather than of a strictly energetic coupling of catabolic and anabolic reactions.
A consideration of possible mechanisms for the oxidative assimilation of pyruvic and lactic acids indicates acetic acid as the most likely starting point for the assimilatory process proper.
Experimental investigations of the mode of acetate breakdown began with studies on the oxidation of glycolic acid. This substance is shown to be an oxidation catalyst in the metabolism of Prototheca zopfii. The exact nature of the catalytic function has not yet been determined.