Steady-state kinetic analysis of human ubiquitin-activating enzyme (E1) using a fluorescently labeled ubiquitin substrate

We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys...

Full description

Saved in:
Bibliographic Details
Published in:Journal of Protein Chemistry 2000-08, Vol.19 (6), p.489-498
Main Authors: Wee, K E, Lai, Z, Auger, K R, Ma, J, Horiuchi, K Y, Dowling, R L, Dougherty, C S, Corman, J I, Wynn, R, Copeland, R A
Format: Article
Language:English
Subjects:
Binding, Competitive
Catalysis
Enzymes
Fluoresceins - chemistry
Fluorescence
Humans
Kinetics
Ligases - chemistry
Ligases - metabolism
Models, Molecular
Molecular biology
Protein Structure, Quaternary
Reaction kinetics
Sodium chloride
Steady state
Substrate Specificity
Substrates
Ubiquitin
Ubiquitin-Activating Enzymes
Ubiquitin-conjugating enzyme
Ubiquitin-Protein Ligases
Ubiquitins - metabolism
Ultracentrifugation
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 of Ub. We demonstrate that Og-Ub is efficiently accepted by Ub-utilizing enzymes, such as the human ubiquitin-activating enzyme (E1). We used this fluorescent substrate to follow the steady-state kinetics of human E1-catalyzed Ub-transfer to the ubiquitin-carrier enzyme Ubc4. In this reaction, E1 uses three substrates: ATP, Ubc4, and Ub. The steady-state kinetics of Og-Ub utilization by E1 is presented. We have also used analytical ultracentrifugation methods to establish that E1 is monomeric under our assay condition (low salt) as well as under physiological condition (150 mM NaCl).
ISSN:0277-8033
1572-3887
1573-4943
DOI:10.1023/A:1026501515450