Kinetic Mechanism of Nicotinamide N-Methyltransferase

Nicotinamide N-methyltransferase (NNMT) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to nicotinamide, pyridine, and other structural analogues. Aberrantly increased NNMT activity results in the depletion of SAM, nicotinamide (NAM), and nicotinamide adenine dinucleotide (NAD⁺); NAM is required for NAD⁺ biosynthesis. SAM depletion impairs the methylation potential of the cell, resulting in hypomethylated histones and an altered epigenetic profile. In addition, decreased NAD⁺ levels negatively affect energy metabolism by disrupting oxidative phosphorylation. Because of its impact on epigenetic states and NAD⁺ levels, NNMT is implicated in cancer, neurodegenerative diseases, and metabolic diseases, making it an appealing target for therapeutic intervention. To gain insights that would guide the design of inhibitors and activity-based probes, we performed detailed kinetic studies of human NNMT. Herein, we report the kinetic mechanism of NNMT. Our initial velocity, product inhibition, and dead-end analogue inhibition studies collectively indicate that NNMT uses a rapid equilibrium ordered mechanism, where NNMT first binds SAM, which is followed by NAM. Methyl transfer occurs, and methylated NAM and S-adenosylhomocysteine are released consecutively.