A NEW PARADIGM FOR BIOCHEMICAL ENERGY COUPLING - SALMONELLA-TYPHIMURIUM NICOTINATE PHOSPHORIBOSYLTRANSFERASE

The pncB gene of Salmonella typhimurium was used to develop an overexp ression system for nicotinate phosphoribosyltransferase (NAPRTase, EC 2.4.2.11), which forms nicotinate mononucleotide (NAMN) and PP(i) from nicotinate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). NAPRT ase hydrolyzes ATP in 1:1 molar stoichiometry to NAMN synthesis. Hydro lysis of ATP alters the ratio of products/substrates for the reaction nicotinate + PRPP half arrow right over half arrow left NAMN + PP(i) f rom its equilibrium value of 0.67 to a steady-state value of 1100. The energy for the maintenance of this ratio must come from ATP hydrolysi s. However, in contrast to other ATP-utilizing enzymes, when all ATP i s hydrolyzed the unfavorable product/substrate ratio collapses. ATP/AD P exchange results suggest that the overall reaction involves a phosph oenzyme (E-P) arising from E. ATP. K(m) values for nicotinate and PRPP each decreased by 200-fold when ATP was present to phosphorylate the enzyme. PP(i) stimulated the ATPase activity of the enzyme to V(max) v alues, suggesting that PP(i) formation during catalysis provides a tri gger for cleavage of the putative E-P in the overall reaction and rege nerates the low affinity form of the enzyme. A model is presented in w hich alternation of high and low affinity forms of NAPRTase provides a ''steady-state'' coupling between ATP hydrolysis and NAMN formation.