Conservation in the Mechanism of Nedd8 Activation by the Human AppBp1-Uba3 Heterodimer

Human Nedd8-activating enzyme AppBp1-Uba3 was purified to apparent homogeneity from erythrocytes. In the presence of [2,8-3H]ATP and 125I-Nedd8, heterodimer rapidly forms a stable stoichiometric ternary complex composed of tightly bound Nedd8 [3H]adenylate and Uba3-125I-Nedd8 thiol ester. Isotope ex...

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Bibliographic Details
Published in:The Journal of biological chemistry 2003-07, Vol.278 (29), p.26823-26830
Main Authors: Bohnsack, Richard N., Haas, Arthur L.
Format: Article
Language:English
Subjects:
Amyloid beta-Protein Precursor - metabolism
Dimerization
DNA-Binding Proteins - chemistry
DNA-Binding Proteins - metabolism
Erythrocytes - metabolism
HeLa Cells
Humans
In Vitro Techniques
Kinetics
Macromolecular Substances
NEDD8 Protein
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Ubiquitin-Activating Enzymes
Ubiquitins - chemistry
Ubiquitins - metabolism
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Summary:Human Nedd8-activating enzyme AppBp1-Uba3 was purified to apparent homogeneity from erythrocytes. In the presence of [2,8-3H]ATP and 125I-Nedd8, heterodimer rapidly forms a stable stoichiometric ternary complex composed of tightly bound Nedd8 [3H]adenylate and Uba3-125I-Nedd8 thiol ester. Isotope exchange kinetics show that the heterodimer follows a pseudo-ordered mechanism with ATP the leading and Nedd8 the trailing substrate. Human AppBp1-Uba3 follows hyperbolic kinetics for HsUbc12 transthiolation with 125I-Nedd8 (kcat = 3.5 ± 0.2 s–1), yielding Km values for ATP (103 ± 12 μm), 125I-Nedd8 (0.95 ± 0.18 μm), and HsUbc12 (43 ± 13 nm) similar to those for ubiquitin activation by Uba1. Wild type 125I-ubiquitin fails to support AppBp1-Uba3 catalyzed activation or HsUbc12 transthiolation. However, modest inhibition of 125I-Nedd8 ternary complex formation by unlabeled ubiquitin suggests a Kd > 300 μm for ubiquitin. Alanine 72 of Nedd8 is a critical specificity determinant for AppBp1-Uba3 binding because 125I-UbR72L undergoes heterodimer-catalyzed hyperbolic HsUbc12 transthiolation and yields Km = 20 ± 9 μm and kcat = 0.9 ± 0.3 s–1. These observations demonstrate remarkable conservation in the mechanism of AppBp1-Uba3 that mirrors its sequence conservation with the Uba1 ubiquitin-activating enzyme.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M303177200