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

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The kinetic analysis of the N -methylation of 4-phenylpyridine by nicotinamide N -methyltransferase: Evidence for a novel mechanism of substrate inhibition. / van Haren, Matthijs J.; Thomas, Martin G.; Sartini, Davide; Barlow, David J.; Ramsden, David B.; Emanuelli, Monica; Klamt, Fábio; Martin, Nathaniel I.; Parsons, Richard B.

In: The International Journal of Biochemistry & Cell Biology, Vol. 98, 01.05.2018, p. 127-136.

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van Haren, Matthijs J. ; Thomas, Martin G. ; Sartini, Davide ; Barlow, David J. ; Ramsden, David B. ; Emanuelli, Monica ; Klamt, Fábio ; Martin, Nathaniel I. ; Parsons, Richard B. / The kinetic analysis of the N -methylation of 4-phenylpyridine by nicotinamide N -methyltransferase: Evidence for a novel mechanism of substrate inhibition. In: The International Journal of Biochemistry & Cell Biology. 2018 ; Vol. 98. pp. 127-136.

Bibtex

@article{011db89517a14fa6a7abaef1c9726ae1,
title = "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 Km for 4-phenylpyridine was similar to that reported for nicotinamide, its kcat of 9.3 × 10−5 ± 2 × 10−5 s−1 and specificity constant, kcat/Km, 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 (<2.5 mM) substrate concentration, 4-phenylpyridine N-methylation was competitively inhibited by dimethylsulphoxide, with a Ki of 34 ± 8 mM. At high (>2.5 mM) substrate concentration, enzyme activity followed substrate inhibition kinetics, with a Ki 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.",
keywords = "enzyme kinetics , neurotoxicity , N-Methylation , substrate inhibition , substrate specificity ",
author = "{van Haren}, {Matthijs J.} and Thomas, {Martin G.} and Davide Sartini and Barlow, {David J.} and Ramsden, {David B.} and Monica Emanuelli and F{\'a}bio Klamt and Martin, {Nathaniel I.} and Parsons, {Richard B.}",
year = "2018",
month = may,
day = "1",
doi = "10.1016/j.biocel.2018.03.010",
language = "English",
volume = "98",
pages = "127--136",
journal = "The International Journal of Biochemistry & Cell Biology",
issn = "1357-2725",
publisher = "Elsevier",

}

RIS

TY - JOUR

T1 - The kinetic analysis of the N -methylation of 4-phenylpyridine by nicotinamide N -methyltransferase:

T2 - Evidence for a novel mechanism of substrate inhibition

AU - van Haren, Matthijs J.

AU - Thomas, Martin G.

AU - Sartini, Davide

AU - Barlow, David J.

AU - Ramsden, David B.

AU - Emanuelli, Monica

AU - Klamt, Fábio

AU - Martin, Nathaniel I.

AU - Parsons, Richard B.

PY - 2018/5/1

Y1 - 2018/5/1

N2 - 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 Km for 4-phenylpyridine was similar to that reported for nicotinamide, its kcat of 9.3 × 10−5 ± 2 × 10−5 s−1 and specificity constant, kcat/Km, 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 (<2.5 mM) substrate concentration, 4-phenylpyridine N-methylation was competitively inhibited by dimethylsulphoxide, with a Ki of 34 ± 8 mM. At high (>2.5 mM) substrate concentration, enzyme activity followed substrate inhibition kinetics, with a Ki 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.

AB - 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 Km for 4-phenylpyridine was similar to that reported for nicotinamide, its kcat of 9.3 × 10−5 ± 2 × 10−5 s−1 and specificity constant, kcat/Km, 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 (<2.5 mM) substrate concentration, 4-phenylpyridine N-methylation was competitively inhibited by dimethylsulphoxide, with a Ki of 34 ± 8 mM. At high (>2.5 mM) substrate concentration, enzyme activity followed substrate inhibition kinetics, with a Ki 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.

KW - enzyme kinetics

KW - neurotoxicity

KW - N-Methylation

KW - substrate inhibition

KW - substrate specificity

U2 - 10.1016/j.biocel.2018.03.010

DO - 10.1016/j.biocel.2018.03.010

M3 - Article

VL - 98

SP - 127

EP - 136

JO - The International Journal of Biochemistry & Cell Biology

JF - The International Journal of Biochemistry & Cell Biology

SN - 1357-2725

ER -