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, 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.

Assay Systems for Profiling Deubiquitinating Activity

“…in vitro deubiquitinating assay Fluorescence Ub-AMC [30][31][32][33]89] Ub-PLA 2 [36,37] Ub-Rho110 [38][39][40]90] FP-based Ub-Lys-TAMRA-Gly [44][45][46]53] FRET di-Ub [50][51][52] Luminescence Ub-AML [34,35] Gel staining Ubiquitin chain [27,32,77] Deubiquitinating assay with mammalian cells…”

“…Ub-AMC is the most frequently used fluorescent ubiquitin substrate whose C-terminal derivative is AMC [30]. This derivative quenches the intrinsic fluorescence but releases fluorescence following hydrolysis by DUBs; fluorescence emission facilitates the real-time monitoring of the sensitivity or activity of DUBs [31]. This monitoring can be performed using 380 nm excitation and 460 nm emission wavelengths.…”