Purification and properties of thiamine pyrophosphokinase from parsely leaf.
Thiamine pyrophosphokinase was purified about 8,000-fold from extracts of parsely leaves. The enzyme, as prepared, was homogenous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme, estimated by gel filtration with Sephadex G-150, was approximately 30,000. In 0.05 M Tris-HCl, the enzymic activity showed a pH optimum over a range of 8 to 9. A least squares analyses of Lineweaver-Burk and Hofstee plots gave Km values of 0.8mM and 0.15mum for ATP and thiamine, respectively. Thiamine homologues and analogues so far tested, except for cetyl thiamine, were all inactive as substrates. The enzyme was specific for ATP and Mg++, although to a lesser extent a combination with other ribonucleoside triphosphates or divalent cations could replace them. SH reagents, such as PCMB, NEM and iodoacetamide, were potent inhibitors of the enzyme. The inhibition was prevented by the addition of dithiothreitol. Inorganic pyrophosphate exhibited striking inhibition. TMP could not replace thiamine as the substrate, whereas it inhibited the TPP formation from thiamine. These findings are consistent with the views that TMP is not directly converted to TPP but after being dephosphorylated by the action of a monoesterase, thiamine is pyrophosphorylated with ATP by thiamine pyrophosphokinase (EC 2.7.6.2) to form TPP and thus give a clear evidence regarding the mechanism of TPP formation in plant tissues.
D000645:Ammonium Sulfate; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009711:Nucleotides / Q000494:pharmacology; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D013831:Thiamine
D009711:Nucleotides; D013439:Sulfhydryl Reagents; D010770:Phosphotransferases; D000645:Ammonium Sulfate; D013831:Thiamine