Abstract
Thymidine phosphorylase (thymidine + P i thymine + 2-deoxy- d-ribose-1-phosphate, EC 2.4.2.4) from Salmonella typhimurium, LT-2 has been purified 257-fold. A molecular weight for the native enzyme of 100,000 ± 10% was determined by Sephadex G-150 filtration. Disc gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate revealed a subunit molecular weight of 47,000, indicating that the enzyme is composed of two subunits. The purified enzyme was found to be specific for the pyrirnidine deoxyribonucleosides, thymidine and deoxyuridine. The Michaelis constants for thymidine, deoxyuridine, phosphate, and arsenate were 2.1 m m, 8.0 m m, 2.3 m m, and 1.3 m m, respectively. Initial velocity and product inhibition studies are consistent with an ordered Bi Bi mechanism, with the nucleoside the first substrate to add to, and the pyrirnidine base the last product to leave, the enzyme surface.
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