Product inhibition kinetics determinations - Substrate interaction affinity and enzymatic kinetics using one quantitative FRET assay.

Autor: Liu Y; Department of Bioengineering, Bourns College of Engineering, Biomedical Science, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA., Zhang F; State Key Laboratory of Oral Diseases, Department of Periodontology, National Clinical Research Center for Oral Diseases, West China, Hospital of Stomatology, Sichuan University, Chengdu, China; Physical Examination Center, West China Hospital, Sichuan University, Chengdu, China., Jiang L; Department of Bioengineering, Bourns College of Engineering, Biomedical Science, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA., Perry JJP; City of Hope Biomedical Research Center, 1218 S. Fifth Avenue, Office 2268 Monrovia, CA 91016, USA., Zhao Z; State Key Laboratory of Oral Diseases, Department of Periodontology, National Clinical Research Center for Oral Diseases, West China, Hospital of Stomatology, Sichuan University, Chengdu, China. Electronic address: zhzhao@scu.edu.cn., Liao J; Department of Bioengineering, Bourns College of Engineering, Biomedical Science, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA; Biomedical Science, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA. Electronic address: jiayu.liao@ucr.edu.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2021 Dec 15; Vol. 193 (Pt B), pp. 1481-1487. Date of Electronic Publication: 2021 Nov 13.
DOI: 10.1016/j.ijbiomac.2021.10.211
Abstrakt: Product inhibition is a common phenomenon during enzyme-catalyzed reactions. Almost all product molecules of an enzyme reaction should have some structural similarities to the substrate, and can thus still have affinities to the active site of the enzyme as product inhibitor. Currently, the characterizations of product inhibition are generally carried out by different methods to determine product binding affinity to the enzyme and the enzyme kinetics parameters, and then these parameters are combined to determine product inhibition. However, due to different sensitivity and variations, kinetics parameters determined from different methods are often not compatible, resulting in not accurate measurement. Here, we report a novel method that determines the two different classes of kinetics parameters, IC 50 and K i (or K D ), K cat and K M , using one single assay method-quantitative FRET(qFRET) assay for characterizing the product inhibition of pre-SUMO1's maturation by its protease SENP1. One method to determine all kinetics parameters provides, for the first time, not only a convenient method to determine all kinetics parameters, but more importantly, a novel approach to combine different measurements with mutually compatible results and errors.
(Copyright © 2021 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
Externí odkaz: