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J. Biol. Chem., Vol. 278, Issue 29, 26823-26830, July 18, 2003

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

Richard N. Bohnsack and Arthur L. Haas 

From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

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 (k_cat = 3.5 ± 0.2 s^–1), yielding K_m 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 K_d > 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 K_m = 20 ± 9 µM and k_cat = 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.

Received for publication, March 27, 2003 , and in revised form, April 28, 2003.

^* This work was supported by United States Public Service Health Grant GM34009 (to A. L. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-8768; Fax: 414-456-6510; E-mail: arthaas{at}mcw.edu.

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