@inbook{4176f361d7d74e888657a2791367ca42,
title = "Competition assay for measuring deubiquitinating enzyme substrate affinity",
abstract = " Assays of the affinity of a deubiquitinating enzyme for substrate, either through binding studies or determination of the Michaelis constant, K M , 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, K i , provides a reliable approximation of the K d 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.",
keywords = "Deubiquitinating enzymes, Enzyme inhibition, Enzyme kinetics, Equilibrium binding, Ubiquitin",
author = "Morgan, {Michael T.} and Cynthia Wolberger",
note = "Publisher Copyright: {\textcopyright} 2018, Springer Science+Business Media, LLC, part of Springer Nature.",
year = "2018",
doi = "10.1007/978-1-4939-8706-1_5",
language = "English (US)",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "59--70",
booktitle = "Methods in Molecular Biology",
}