Mechanism of the Enzymatic Phosphorylation of Thiamin

Abstract

WE have previously demonstrated that thiamin pyrophosphate may be synthesized from the free vitamin by the action of enzymes present in alkaline washed brewer's bottom yeast, provided that hexose-diphosphate and certain factors present in boiled aqueous extracts of animal tissues are present^1,3. Synthesis under these conditions was completely inhibited by 0·005 M. sodium iodoacetate, but was relatively unaffected by 0·04 M. sodium fluoride. More recently³, we have demonstrated that cozymase and acetaldehyde may be substituted for the boiled tissue extract with no change in the degree of synthesis or of the iodoacetate inhibition. Phosphoglyceric acid may also be substituted for the boiled tissue extract, but under these conditions iodoacetate fails to affect the synthesis while fluoride causes complete inhibition³. Using specially prepared alkaline washed yeast, we have found that cocarboxylase synthesis with phosphoglyceric acid is considerably diminished. It may be restored, however, by the addition of catalytic quantities of adenylic acid. As a result of these experiments we have proposed the reaction