Characterization of the enzymatic activity of the rhythmic human NADP(H) phosphatase, Nocturnin

NAD(P)(H) is an essential cofactor for metabolic processes and a protector against oxidative stress. Phosphorylation of NAD(H) is completed by NAD kinase while dephosphorylation is performed by Nocturnin (NOCT). Because the phosphatase activity of NOCT is a rather new discovery, its enzymatic mechanism has not been reported. In this study, we use classical steady-state kinetics and pH-rate profiles of site-directed mutants in the NOCT active-site to deduce the essential residues for catalytic function for dephosphorylation of NADP(H). The pH-rate profile is bell shaped which supports that NOCT performs acid-base catalysis. Site-directed mutagenesis showed N149 is important for coordinating a deprotonated nearby residue, which is likely D324, and activates a nearby water to act as the nucleophile. After hydrolysis is performed, protonated H286 donates the proton to the leaving group. Finally, NAD(H) and inorganic phosphate is released. Since NOCT has been implicated in many important metabolic and stress response-related processes, understanding the mechanism is essential for the development of pharmacological modulators of NOCT activity.