Abstract
Thiamin pyrophosphokinase purified to homogeneity from pig brain showed following properties. 1) The enzyme required Mg^<2+>, Co^<2+> or Zn^<2+> as a cofactor and ATP or UTP as a cosubstrate for its activity. 2) The pH optimum was between pH 6 and 7,and the Km values for thiamin and ATP were 0.43μM and 0.78 mM, respectively. 3) The reaction was inhibited by several nucleotides among which GTP, GDP, GMP and IMP showed remarkable inhibitions by competing with ATP, and Ki values for the nucleotides were 3.8,1.5,20 and 34μM, respectively. 4) Thiamin analogues such as pyrithiamin, 2-northiamin and chloroethylthiamin were competitive inhibitors for thiamin with Ki values of 0.85,5.8 and 27μM, respectively, whereas TMP, TDP and thiamin sulfate showed noncompetitive inhibitions with Ki values of 17,0.67 and 6.0μM, respectively. 5) Neuroactive amines such as serotonin and dopamine acted as weak inhibitors of the enzyme and the inhibitions appeared to be competitive with respect to thiamin. 6) The purified enzyme was reversiblly inactivated by SH reagents such as PCMB and NEM suggesting that it is an SH enzyme.
