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

Yan Liu; Fan Zhang; Ling Jiang; J Jefferson P Perry; Zhihe Zhao; Jiayu Liao

doi:10.1016/j.ijbiomac.2021.10.211

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

Int J Biol Macromol. 2021 Dec 15;193(Pt B):1481-1487. doi: 10.1016/j.ijbiomac.2021.10.211. Epub 2021 Nov 13.

Authors

Yan Liu¹, Fan Zhang², Ling Jiang¹, J Jefferson P Perry³, Zhihe Zhao⁴, Jiayu Liao⁵

Affiliations

¹ Department of Bioengineering, Bourns College of Engineering, Biomedical Science, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA.
² 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.
³ City of Hope Biomedical Research Center, 1218 S. Fifth Avenue, Office 2268 Monrovia, CA 91016, USA.
⁴ 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.
⁵ 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.

PMID: 34780893
DOI: 10.1016/j.ijbiomac.2021.10.211

Abstract

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, IC₅₀ 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.

Keywords: Enzyme kinetics; Product inhibition; Protein interaction affinity; Quantitative FRET(qFRET) assay; SUMOylation.

MeSH terms

Catalytic Domain
Cysteine Endopeptidases / metabolism*
Enzyme Assays / methods
Fluorescence Resonance Energy Transfer / methods
Kinetics
Peptide Hydrolases / metabolism*
Substrate Specificity

Substances

Peptide Hydrolases
Cysteine Endopeptidases