The kinetic analysis of the N-methylation of 4-phenylpyridine by nicotinamide N-methyltransferase: Evidence for a novel mechanism of substrate inhibition

Abstract The N -methylation of 4-phenylpyridine produces the neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+). We investigated the kinetics of 4-phenylpyridine N -methylation by nicotinamide N- methyltransferase (NNMT) and its effect upon 4-phenylpyridine toxicity in vitro . Human recombinant NNMT possessed 4-phenylpyridine N -methyltransferase activity, with a specific activity of 1.7 ± 0.03 nmol MPP+ produced/h/mg NNMT. Although the K m for 4-phenylpyridine was similar to that reported for nicotinamide, its k cat of 9.3 × 10 −5  ± 2 × 10 −5  s −1 and specificity constant, k cat / K m , of 0.8 ± 0.8 s −1  M −1 were less than 0.15% of the respective values for nicotinamide, demonstrating that 4-phenylpyridine is a poor substrate for NNMT. At low ( N -methylation was competitively inhibited by dimethylsulphoxide, with a K i of 34 ± 8 mM. At high (>2.5 mM) substrate concentration, enzyme activity followed substrate inhibition kinetics, with a K i of 4 ± 1 mM. In silico molecular docking suggested that 4-phenylpyridine binds to the active site of NNMT in two non-redundant poses, one a substrate binding mode and the other an inhibitory mode. Finally, the expression of NNMT in the SH-SY5Y cell-line had no effect cell death, viability, ATP content or mitochondrial membrane potential. These data demonstrate that 4-phenylpyridine N -methylation by NNMT is unlikely to serve as a source of MPP+. The possibility for competitive inhibition by dimethylsulphoxide should be considered in NNMT-based drug discovery studies. The potential for 4-phenylpyridine to bind to the active site in two binding orientations using the same active site residues is a novel mechanism of substrate inhibition.