Enzyme system involved in the synthesis of thiamin triphosphate. I. Purification and characterization of protein-bound thiamin diphosphate: ATP phosphoryltransferase.
An enzyme system catalyzing the synthesis of thiamin triphosphate consists of an enzyme (protein-bound thiamin diphosphate:ATP phosphoryltransferase), thiamin diphosphate bound to a macromolecule as substrate, ATP, Mg2+, and a low molecular weight cofactor. This system was established by combining a purified enzyme and an essentially pure, macromolecule-bound substrate prepared from rat livers. This macromolecule was found to be a protein, and the transphosphorylation of thiamin diphosphate to thiamin triphosphate with ATP and enzyme was shown to occur on this macromolecule which binds thiamin diphosphate. Free thiamin, thiamin monophosphate, thiamin diphosphate, and thiamin triphosphate have no effect on this reaction. Thus, the overall reaction is: thiamin diphosphate-protein + ATP in equilibrium thiamin triphosphate-protein + ADP. So-called thiamin diphosphate:ATP phosphoryltransferase (EC 2.7.4.15) activity was not detected in rat brain or liver. The enzyme was extracted from acetone powder of a crude mitochondrial fraction of bovine brain cortex and purified to homogeneity with a 0.6% yield after DEAE-cellulose chromatography, a first gel filtration, hydroxylapatite chromatography, chromatofocusing, and a second gel filtration. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its molecular weight was estimated to be 103,000. The pH optimum was 7.5, and the Km was determined to be 6 X 10(-4) M for ATP. ATP was found to be the most effective phosphate donor among the nucleoside triphosphates. Amino acid analysis of the purified enzyme revealed an abundance of glutaminyl, glutamyl, and aspartyl residues. Sulfhydryl reagents inhibited the enzyme reaction. Metals such as Fe2+, Zn2+, Pb2+, and Cu2+ strongly inhibited the activity. The enzyme was unstable, and glycerol (20%) and dithiothreitol (1.0 mM) were found to preserve the enzyme activity.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002413:Cations, Divalent; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor); D051381:Rats; D011919:Rats, Inbred Strains; D013831:Thiamine / Q000031:analogs & derivatives*; D013838:Thiamine Triphosphate / Q000096:biosynthesis* / Q000302:isolation & purification
D000596:Amino Acids; D002413:Cations, Divalent; D013838:Thiamine Triphosphate; D010770:Phosphotransferases; D017856:Phosphotransferases (Phosphate Group Acceptor); C022733:thiamin-diphosphate kinase; D013831:Thiamine