Competition assay for measuring deubiquitinating enzyme substrate affinity

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Assays of the affinity of a deubiquitinating enzyme for substrate, either through binding studies or determination of the Michaelis constant, KM, can shed light on substrate selectivity and the effects of mutations on substrate interactions. The difficulty in generating sufficient quantities of ubiquitinated substrate frequently presents a barrier to these studies. We describe here an alternative approach that can be used in cases where non-hydrolyzable, chemically ubiquitinated substrate analogs can be more readily generated. The substrate analog can be utilized as a competitive inhibitor in kinetics experiments monitoring cleavage of ubiquitin-AMC (Ub-AMC) by the deubiquitinating enzyme. The resulting inhibitory constant, Ki, provides a reliable approximation of the Kd for ubiquitinated substrate. We show how this approach can be used to assay the affinity of the yeast SAGA DUB module for nucleosomes containing monoubiquitinated H2B.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages59-70
Number of pages12
DOIs
Publication statusPublished - Jan 1 2018

Publication series

NameMethods in Molecular Biology
Volume1844
ISSN (Print)1064-3745

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Keywords

  • Deubiquitinating enzymes
  • Enzyme inhibition
  • Enzyme kinetics
  • Equilibrium binding
  • Ubiquitin

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Morgan, M., & Wolberger, C. (2018). Competition assay for measuring deubiquitinating enzyme substrate affinity. In Methods in Molecular Biology (pp. 59-70). (Methods in Molecular Biology; Vol. 1844). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8706-1_5