Application of a coupled enzyme assay to characterize nicotinamide riboside kinases

Nicotinamide adenine dinucleotide (NAD) has versatile functions in all cells. Besides its involvement in energy metabolism it serves important functions as substrate in a variety of cellular signaling reactions.  Although the redox reactions do not result in a net consumption of the molecule, the signaling reactions are accompanied by cleavage of NAD+. Therefore, continuous resynthesis of pyridine nucleotides is vital. The biosynthesis of NAD in humans can proceed via four different routes: a de novo synthesis pathway from tryptophan, a pathway from the vitamin B3 precursor nicotinic acid (NA) or nicotinamide (Nam), or phosphorylation of nicotinamide riboside (NR), a newly discovered vitamin precursor. NR Kinases (Nrks) are nucleoside kinases that are supposed to preferentially generate nicotinamide mononucleotide (NMN) from NR using adenosine triphosphate (ATP) as phosphoryl donor. They have only recently been identified, and two isoforms were shown to exist in humans. The physiological roles and substrate specificities of these enzymes have so far been studied only poorly, partly because of a lack of convenient enzyme assays. High-performance liquid chromatography (HPLC) analyses have been used, for example, to test pyrimidine nucleosides as potential alternative substrates of Nrks. The commonly used method to measure Nrk activity includes, besides NR and ATP as substrates, NMN adenylyl transferase (NMNAT) and alcohol dehydrogenase (ADH).

Based on these considerations the author has developed an alternative photometric assay that eliminates these drawbacks and applied it to measure kinetic parameters of human recombinant Nrk1 and Nrk2. The system makes use of the fact that a “side” product of the Nrk-catalyzed reaction is adenosine diphosphate (ADP). Therefore, a standard assay for adenosine triphosphatase (ATPase) activity can be used to measure Nrk activity. Advantageously, ATP is regenerated from ADP and phosphoenolpyruvate by pyruvate kinase (PK). The other product, pyruvate, is reduced to lactate by lactate dehydrogenase (LDH). Lactate formation is accompanied by oxidation of (added) NADH to NAD+, which is easily measured spectrophotometrically. Besides the ease of the measurement(PK/LDH is commercially available as a ready mix for ATPase determinations), this system also does not require the authentic nucleoside (NR) because NMN does not need to be formed for detection of enzyme activity. Therefore, the nucleoside specificity and according kinetic parameters of mononucleotide formation can be established conveniently using this assay. In addition, the PK/LDH-coupled assay enables an accuratemeasurement of the affinity of Nrk for ATP because it can be measured under steady-state conditions. That is, each molecule of ATP used is restored by PK from the added phosphoenolpyruvate and the ADP generated in the Nrk reaction. Therefore, the ATP concentration remains unchanged during the assay. The consumption of each molecule of ATP by Nrk is detected as one molecule NADH oxidized by LDH when pyruvate is converted to lactate. At low micromolar concentrations, the spectrophotometric measurementof NADH is not very accurate due to the comparably low extinction coefficient. However, the reaction can be continued over a long period of time (depending on the Nrk activity used), and the linear decrease of the NADH concentration correlates directly with the Nrk activity. Therefore, low KM values for ATP can be established with confidence. In fact, because guanosine diphos-phate (GDP) is as good a substrate for PK as ADP, the kinetics of Nrks with regard to guanosine triphosphate (GTP) as phosphoryl donor can be determined as well. Finally, owing to the recycling nature of the assay, low Nrk activities can be measured by simply extending the reaction time.

A disadvantage of this systemmay consist in the problemthat in crude cell lysates, for example, there are ATPases that do not require a second substrate. Therefore, to measure Nrk activity in lysates, it would be necessary to subtract the unspecific ATPase activity (measured in the absence of NR) fromthatmeasuredwhen NR is present. Obviously, alternative substrates, such as pyrimidine nucleosides (for which other kinases exist), cannot be used so long as the presence of contaminating activities cannot be ruled out. Therefore, the suitability of this assay will need to be established for the specific biological sample of interest (e.g., tissue or cell lysates) and depends

on the balance of Nrk activity and overall ATPase activity.

To generate NR, the author usually use NAD+ as startingmaterial (rather than the far more expensive NMN), cleave it into NMN and AMP by treatment with phosphodiesterase I from snake venom, and then dephosphorylate the mononucleotides to nicotinamide riboside and adenosine, respectively, by alkaline phosphatase treatment. The resultant nucleosides are easily separated by reverse-phase HPLC. The fractions containing NR are pooled, dried in aliquots by lyophilization, and stored at -20 oC. To simplify the procedure, the author routinely add both phosphodiesterase and phosphatase simultaneously and leave the mixture at room temperature overnight. The reaction mixture should be buffered sufficiently to avoid acidification due to proton release during the course of the reaction.




Christian Dölle, Analytical Biochemistry 385 (2009) 377-379