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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Published on: July 25, 2019

Competition Assay for Measuring Deubiquitinating Enzyme Substrate Affinity.

Michael T Morgan¹, Cynthia Wolberger²

¹Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Methods in Molecular Biology (Clifton, N.J.)

|September 23, 2018

Summary

Researchers developed a new method to measure deubiquitinating enzyme affinity using non-hydrolyzable substrate analogs. This approach overcomes challenges in generating ubiquitinated substrates for enzyme kinetics and binding studies.

Keywords:

Deubiquitinating enzymes Enzyme inhibition Enzyme kinetics Equilibrium binding Ubiquitin

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Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Published on: May 10, 2015

Area of Science:

Biochemistry
Enzymology
Molecular Biology

Background:

Assessing deubiquitinating enzyme (DUB) affinity for substrates is crucial for understanding enzyme selectivity and mutation effects.
Generating sufficient ubiquitinated substrates for traditional binding or kinetic assays (K_M) is often a significant challenge.

Purpose of the Study:

To present an alternative method for assaying DUB-substrate affinity when ubiquitinated substrates are difficult to produce.
To validate the use of non-hydrolyzable substrate analogs as competitive inhibitors in enzyme kinetics.

Main Methods:

Utilized chemically synthesized, non-hydrolyzable ubiquitinated substrate analogs.
Employed competitive inhibition kinetics assays monitoring the cleavage of ubiquitin-AMC (Ub-AMC).
Measured the inhibitory constant (K_i) of the substrate analog.

Main Results:

The inhibitory constant (K_i) derived from competitive inhibition assays reliably approximates the dissociation constant (K_d) of the actual ubiquitinated substrate.
Demonstrated the successful application of this method to determine the affinity of the yeast SAGA DUB module for nucleosomes with monoubiquitinated H2B.

Conclusions:

This novel approach provides a practical alternative for DUB affinity studies, circumventing the need for large quantities of ubiquitinated substrates.
The method enables robust characterization of DUB-substrate interactions, including those involving complex substrates like ubiquitinated nucleosomes.