New insights into the catalytic mechanism of human glycine N‐acyltransferase

Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi‐substrate kinetic parameters of a recombinant human glycine N‐acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colo...

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Bibliographic Details
Published in:Journal of biochemical and molecular toxicology 2017-11, Vol.31 (11), p.n/a
Main Authors: der Sluis, Rencia, Ungerer, Vida, Nortje, Carla, Dijk, Alberdina, Erasmus, Elardus
Format: Article
Language:English
Subjects:
Acyl Coenzyme A - metabolism
Acyltransferases - chemistry
Acyltransferases - genetics
Acyltransferases - metabolism
Allosteric properties
Catalysis
Chromatography, High Pressure Liquid - methods
Colorimetry
Colorimetry - methods
Conjugation
Enzyme kinetics
enzyme mechanism
Glycine
Glycine - metabolism
Glycine N-acyltransferase
High-performance liquid chromatography
Hippurates - analysis
Hippurates - metabolism
Hippuric acid
HPLC–ESI‐MS/MS
human
Humans
Kinetics
Liquid chromatography
Metabolic pathways
Reaction mechanisms
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Solvents - chemistry
Spectrometry, Mass, Electrospray Ionization - methods
Substrate inhibition
Tandem Mass Spectrometry - methods
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Summary:Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi‐substrate kinetic parameters of a recombinant human glycine N‐acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC–ESI‐MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi–Bi and/or ping‐pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl‐CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis–Menten reaction mechanism.
ISSN:1095-6670
1099-0461
DOI:10.1002/jbt.21963