An auxiliary enzyme is an enzyme added to a coupled assay to convert a product or intermediate of the primary reaction into a measurable signal. The primary enzyme generates the chemical species being followed, while one or more supporting reactions connect its formation to a detectable change [1]. Enzyme-based measurements relate the measured signal to the amount or turnover of a particular metabolite through the stoichiometry of the coupled reactions [2]. In an ADP-linked assay, pyruvate kinase converts ADP and phosphoenolpyruvate into ATP and pyruvate, and lactate dehydrogenase couples pyruvate reduction to NADH oxidation. Other coupled systems use sequential carbohydrate-converting enzymes to connect the target reaction with a spectrophotometric readout [3][4]. Sufficient auxiliary activity allows the signal to track the primary reaction without becoming constrained by the supporting conversion steps.
- coupled enzyme assay
- coupling enzyme
- reaction kinetics
- signal generation
Enzyme function and inhibition·Molecular Biology·Biochemistry, Genetics and Molecular Biology·Life Sciences
References
- Jean-Philippe Goddard; Jean-Louis Reymond; Recent advances in enzyme assays. Trends Biotechnol. 2004, 22, 363-370. [CrossRef]
- Wenyun Lu; Xiaoyang Su; Matthias S. Klein; Ian A. Lewis; Oliver Fiehn; Joshua D. Rabinowitz; Metabolite Measurement: Pitfalls to Avoid and Practices to Follow. Annu. Rev. Biochem. 2017, 86, 277-304. [CrossRef]
- Jayodita C Sanghvi; Sergi Regot; Silvia Carrasco; Jonathan R Karr; Miriam V Gutschow; Benjamin Bolival; Markus W Covert; Accelerated discovery via a whole-cell model. Nat. Methods 2013, 10, 1192-1195. [CrossRef]
- Bradley A. Webb; Farhad Forouhar; Fu-En Szu; Jayaraman Seetharaman; Liang Tong; Diane L. Barber; Structures of human phosphofructokinase-1 and atomic basis of cancer-associated mutations. Nature 2015, 523, 111-114. [CrossRef]