Conversion of a cosubstrate to an inhibitor: phosphorylation mutants of nicotinic acid phosphoribosyltransferase.

Abstract

Nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11) forms nicotinic acid mononucleotide (NAMN) and PPi from 5-phosphoribosyl 1-pyrophosphate (PRPP) and nicotinic acid (NA). The Vmax NAMN synthesis activity of the Salmonella typhimurium enzyme is stimulated about 10-fold by ATP, which, when present, is hydrolyzed to ADP and Pi in 1:1 stoichiometry with NAMN formed. The overall NAPRTase reaction involves phosphorylation of a low-affinity form of the enzyme by ATP, followed by generation of a high-affinity form of the enzyme, which then binds substrates and produces NAMN. Hydrolysis of E-P then regenerates the low-affinity form of the enzyme with subsequent release of products. Our earlier studies [Gross, J., Rajavel, M., Segura, E., and Grubmeyer, C. (1996) Biochemistry 35, 3917-3924] have shown that His-219 becomes phosphorylated in the N1 (pi) position by ATP. Here, we have mutated His-219 to glutamate and asparagine and determined the properties of the purified mutant enzymes. The mutant NAPRTases fail to carry out ATPase, autophosphorylation, or ADP/ATP exchanges seen with wild-type (WT) enzyme. The mutants do catalyze the slow formation of NAMN in the absence of ATP with rates and KM values similar to those of WT. In striking contrast to WT, NAMN formation by the mutant enzymes is competitively inhibited by ATP. Thus, the NAMN synthesis reaction may occur at a site overlapping that for ATP. Previous studies suggest that the yeast NAPRTase does not catalyze NAMN synthesis in the absence of ATP. We have cloned, overexpressed, and purified the yeast enzyme and report its kinetic properties, which are similar to those of the bacterial enzyme.