The applicants have synthesized and characterized non-hydrolyzable C-glycoside analogs of 5-phosphorylribose 1-alpha-diphosphate (PRPP) and determined that it is a competent competitive inhibitor of orotate, hypoxanthine-guanine (H-G) and glutamine:amido phosphoribosyltransferases (PRTases) and that they cannot serve as substrates. They propose to use these compounds to form dead-end ternary complexes of PRTases, previously impossible experiments since no analogs were available that could resist cleavage at the anomeric carbon. For example, the number of bound Mg2+ and Mn2+ ions will be determined by forming the holo complex whose stoichiometry will be determined by neutron activation analysis. The resumed transition in kinetic mechanism of yeast orotate PRTase from ordered sequential to ping pong as [Mg2+] is raised will also be investigated by inversion transfer experiments using 31P NMR and by equilibrium isotope exchange of 32PPi---32P-PRPP; these proposed studies would characterize the dynamics between PRPP and diphosphate in the first """"""""half-reaction"""""""" of orotate PRTase that might bind a 5-phosphorylribosyl oxocarbocation intermediate. They propose a mechanistic scheme for this enzyme whereby the apparent transition in kinetic mechanism results only from a competition in rate between the release of PPi and binding of orotate. They propose an evaluation of the likely role of a putative oxocarbocation intermediate along the reaction coordinate of orotate PRTase (and others) by testing various analogs of the likely intermediate on the enzyme. These compounds are derivatives of 5-O-phosphoiminoribose, which contain both positive charge and pi character at the """"""""reactive center"""""""". As a full complement to these studies they also propose that new bisubstrate analogs be evaluated to test the idea that an Sn2-type ternary complex exists at or near the transition-state. A similar example for purine nucleoside phosphorylase will also be investigated.
| Witte, J F; Tsou, R; McClard, R W (1999) Cloning, overproduction, and purification of native and mutant recombinant yeast orotate phosphoribosyltransferase and the demonstration from magnetization inversion transfer that a proposed oxocarbocation intermediate does not have a kinetic lifetime. Arch Biochem Biophys 361:106-12 |
