Abstract
Enzymatic synthesis of cocarboxylase in rat liver preparation was studied. For this reaction the enzyme requires thiamine as a phosphate acceptor, ATP as a phosphate donor, and divalent cation(s), Mg++ or Mn++, as an activator. Among the adenine nucleotides tested only ATP could serve as a direct substrate and other nucleotides, such as ADP and A5P, did not act as a phosphate donor but behaved as an inhibitor. TMP did not act as a phosphate acceptor but behaved also as an inhibitor. The effectiveness of ADP and A5P+PEP in the crude system may be interpreted as the result of intermediary formation of ATP brought about by other enzymes. Thus, it could be concluded that the only possible mechanism for the reaction is a one-step transpyrophosphorylation. The reaction could not be reversed easily starting from TDP and A5P. The Michaelis constants for thiamine and ATP were calculated as 4.5×10-7 M and 8.3×10-4 M, respectively. The enzyme was inhibited by ethylenediamine tetraacetate.
The author express his sincere thanks to Prof. N. Shimazono for guidance and encouragement during the course of this investigation.
