The kinetic analysis of the N-methylation of 4-phenylpyridine by nicotinamide N-methyltransferase: Evidence for a novel mechanism of substrate inhibition.
Int J Biochem Cell Biol
; 98: 127-136, 2018 05.
Article
in En
| MEDLINE
| ID: mdl-29549048
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⯱â¯0.03â¯nmol MPP+ produced/h/mg NNMT. Although the Km for 4-phenylpyridine was similar to that reported for nicotinamide, its kcat of 9.3â¯×â¯10-5⯱â¯2â¯×â¯10-5â¯s-1 and specificity constant, kcat/Km, of 0.8⯱â¯0.8â¯s-1â¯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 (<2.5â¯mM) substrate concentration, 4-phenylpyridine N-methylation was competitively inhibited by dimethylsulphoxide, with a Ki of 34⯱â¯8â¯mM. At high (>2.5â¯mM) substrate concentration, enzyme activity followed substrate inhibition kinetics, with a Ki of 4⯱â¯1â¯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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Pyridines
/
Protein Processing, Post-Translational
/
Nicotinamide N-Methyltransferase
/
Neuroblastoma
Limits:
Humans
Language:
En
Journal:
Int J Biochem Cell Biol
Journal subject:
BIOQUIMICA
Year:
2018
Document type:
Article
Affiliation country:
Netherlands