Thiamine monophosphate kinase, which catalyzes the formation of thiamine pyrophosphate (TPP) from thiamine monophosphate in the presence of ATP and Mg²⁺, has been purified 60-155 fold from crude extracts of E. coli K12.
The pH optimum of the reaction was around 8.0 and the apparent K_m values for thiamine monophosphate and ATP were l.l×lO^-6M and 2.7×lO^-4_M, respectively. Among the univalent cations tested, K⁺, NH₄⁺, and Rb⁺ markedly activated the enzyme activity, whereas Li⁺, Na⁺, and Cs⁺ antagonized the stimulatory effect of K⁺. The enzyme was inhibited by PCMB and the inhibition by PCMB was reversed by addition of 2-mercaptoethanol. The pathway of TPP synthesis is discussed.