Application of a coupled enzyme assay to characterize nicotinamide riboside kinases.
The recently identified nicotinamide riboside kinases (Nrks) constitute a distinct pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis. Here we present the combination of an established optical adenosine triphosphatase (ATPase) test, the pyruvate kinase/lactate dehydrogenase system, with the Nrk-catalyzed reaction to determine kinetic properties of these enzymes, in particular affinities for ATP. The assay allows variation of both nucleoside and phosphate donor substrates, thereby providing major advantages for the characterization of these enzymes. We confirm previously established kinetic parameters and identify differences in substrate selectivity between the two human Nrk isoforms. The proposed assay is inexpensive and may be applied for high-throughput screening.
Dölle
Christian
C
Department of Molecular Biology, University of Bergen, Bergen, Norway.
Ziegler
Mathias
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000378:metabolism; D012107:Research Design; D013379:Substrate Specificity
D010710:Phosphates; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D017853:Phosphotransferases (Alcohol Group Acceptor); C061783:nicotinamide riboside kinase; D011770:Pyruvate Kinase; D000251:Adenosine Triphosphatases