title: Steady-state kinetic characterization of sesquiterpene synthases by gas chromatography-mass spectroscopy.
abstract: Sesquiterpene synthases produce a wide variety of structurally diverse hydrocarbon products from a single substrate: farnesyl pyrophosphate. Each enzyme will often produce a multitude of products for which the kinetic efficiency is traditionally measured using a radioactivity assay. Here, we introduce a gas chromatography-mass spectroscopy-based assay to measure the formation of a single abundant product from which the kinetic parameters of the enzyme in question can be elucidated. We present an accounting of experimental components and considerations, such as solution conditions and instrument parameters, necessary to perform a standardized vial assay experiment. Further, we outline pilot experiments to establish analyte quantification and the linear range of enzyme concentration versus reaction velocity. Finally, we describe a protocol for a steady-state kinetics experiment, and the processing of experimental data to produce a Michaelis-Menten plot enabling one to derive kinetic parameters.
authors.affiliation: Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich, United Kingdom.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Garrett
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Morris
authors.forename: Paul E
authors.initials: PE
authors.lastname: O'Maille
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D002138:Calibration; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods / Q000592:standards; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods* / Q000592:standards; D007700:Kinetics; D016014:Linear Models; D010865:Pilot Projects; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012717:Sesquiterpenes / Q000378:metabolism*; D012996:Solutions / Q000378:metabolism; D013696:Temperature
chemical_list: D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D012996:Solutions; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase