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.

Manipulation of a Nuclear NAD+ Salvage Pathway Delays Aging without Altering Steady-state NAD+ Levels

“…Our findings show that several SIR2-dependent processes can be enhanced by manipulation of the NAD ϩ salvage pathway in yeast, and this may hold true for higher organisms. We have identified NPT1 homologs in every genome we have examined, and all possess a highly conserved region around a histidine residue that, in Salmonella, greatly stimulates catalysis when phosphorylated (67). This mode of regulation may permit the design of mutations or small molecules that increase Npt1 activity.…”

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…Also, the coupling of ATP hydrolysis to the phosphoribosyltransferase reaction causes a change in the equilibrium value, from 0.67 to 1,100 (253)(254)(255). Mutational replacement of His219 completely abrogates phosphorylation and stimulation by ATP (256). Mammalian organisms such as mice and humans contain yet a different pyridine phosphoribosyltransferase, nicotinamide phosphoribosyltransferase (nicotinamide-D-ribonucleotide:diphosphate phosphor-␣-D-ribosyltransferase; EC 2.4.2.12): nicotinamide ϩ PRPP ¡ 5=-phospho-␤-D-ribosyl-nicotinamide (nicotinamide mononucleotide, NMN) ϩ PP i (257,258).…”

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

“…For example, silencing and replicative aging defects emerge in an npt1D mutant, where NAD + levels are reduced by approximately two-to threefold (Lin et al 2000;Smith et al 2000). Npt1 is a nicotinic acid phosphoribosyltransferase that converts nicotinic acid (NA) into NaMN, the rate-limiting step of the Preiss-Handler NAD + salvage pathway (Rajavel et al 1998). Npt1 and other enzymes of the salvage pathway are enriched in the nucleus Sandmeier et al 2002a), suggesting that there could be a nuclear flux or pool of NAD + that is critical for maintaining nuclear Sir2 functions (see below).…”