ATP-THIAMINE PYROPHOSPHOTRANSFERASE . PURIFICATION AND REACTION MECHANISM STUDY

ATP:thiamine pyrophosphotransferase. Purification and reaction mechanism study 1. 1. ATP:thiamine pyrophosphotransferase (EC 2.7.6.2) from baker's yeast has been purified 200-fold. 2. 2. The enzyme SH groups are not very reactive. The kinase is not completely inhibited by p-chloromercuribenzoate, N-ethylmaleimide and 5,5′-dithio-bis-z-nitrobenzoic acid even at high concentrations. Iodoacetic acid and iodoacetamide are unreactive. 3. 3. The enzyme is fully inhibited by 2,4,6-trinitrobenzene sulfonic acid and strongly by 5-dimethylaminonaphthalane sulfonyl chloride and by maleic anhydride. 4. 4. Some kinetic aspects of the reaction have been examined. The double reciprocal plots of initial velocity versus thiamine concentration at different levels of ATP concentration give a family of parallel lines. In agreement with this result, plotting i/velocity versus i/concentration of ATP for some different ATP/thiamine ratios yields straight lines intercepting on the ordinate axis at the same point. The results of kinetic studies are consistent with a ping-pong" reaction mechanism. © 1969."