Isotopically labeled flavoenzymes and their uses in probing reaction mechanisms.
作者： Andreea I. Iorgu , Matthew J. Cliff , Jonathan P. Waltho , Nigel S. Scrutton , Sam Hay
DOI: 10.1016/BS.MIE.2019.03.009
关键词:
摘要: Abstract The incorporation of stable isotopes into proteins is beneficial or essential for a range experiments, including NMR, neutron scattering and reflectometry, proteomic mass spectrometry, vibrational spectroscopy “heavy” enzyme kinetic isotope effect (KIE) measurements. Here, we present detailed protocols the isotopic labeling pentaerythritol tetranitrate reductase (PETNR) via recombinant expression in E. coli. PETNR an ene-reductase belonging to Old Yellow Enzyme (OYE) family flavoenzymes, regarded as model system studying hydride transfer reactions. Included discussion how efficient back-exchange amide protons protein core can be achieved intrinsic flavin mononucleotide (FMN) cofactor exchanged, allowing production isotopologues with differentially labeled cofactor. In addition thorough description strategies, briefly exemplify data analysis interpretation KIEs performed.
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