THYROID PURINE NUCLEOSIDE PHOSPHORYLASE .2. KINETIC-MODEL BY ALTERNATE SUBSTRATE AND INHIBITION STUDIES

Nucleoside analog inhibition studies have been conducted on thyroidal purine nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) which catalyzed an ordered bi-bi type mechanism where the first substrate is inorganic phosphate and the last product is ribose 1-phosphate. Heterocyclic- and carbohydrate-modified nucleoside inhibitors demostrate mixed type inhibition suggesting such analogs show an affinity (Ki) for the freeze enzyme. A kinetic model is proposed which supports the observed inhibition patterns. These studies together with alternate substrate studies indicate that nucleoside binding requires a functional group capable of hydrogen bonding at the 6-position of the purine ring and that the orientation of the bound substrate may be syn. Proper geometry of the phosphate is dependent upon the 3′-substituent to be orientated below the furanose ring. The 5′-hydroxyl group is required for substrate activity. The proposed rate limiting step of the phosphorylase mechanism is the enzymatic protonation of the 7-N position of the nucleoside. © 1979.