Abstract

Pig brain thiamin pyrophosphokinase (TPK) (ATP: thiamin pyrophosphotransferase, EC 2.7.6.2) 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.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

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