Pig brain thiamin pyrophosphokinase (TPK) (ATP: thiamin pyrophosphotransferase, EC 188.8.131.52) was purified 260-fold over extracts of brain acetone powder. The purification was followed using a direct, radiometric assay. As determined in an analytical ultracentrifuge and by disc-gel electrophoresis, the most highly purified enzyme preparations were not homogeneous. The purified enzyme has a broad pH optimum extending from 8.3 to 9.3 in phosphate-glycylglycine buffer. It has an isoelectric point at about pH 4.2. For optimal enzymatic activity, the ratio of magnesium to ATP must be 0.6. Any deviation from this ratio causes a rapid drop in enzymatic activity. A study of the kinetics of the reaction revealed that the enzyme functions via a "ping-pong" mechanism. Two dissociation constants for ATP and one for thiamin were evaluated. Pyrithiamin, oxythiamin, butylthiamin, and ethylthiamin were competitive inhibitors of thiamin.
College and Department
Chemistry and Biochemistry
BYU ScholarsArchive Citation
Peterson, James Weldon, "Studies of pig brain thiamin pyrophosphokinase" (1971). Theses and Dissertations. 8333.