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 [sup 125]I-Nedd8, heterodimer rapidly forms a stable stoichiometric ternary complex composed of tightly bound Nedd8 [³H]adenylate and Uba3-[sup 125]I-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 [sup 125]I-Nedd8 (k[sub cat] = 3.5 ± 0.2 s[sup -1] yielding K[sub m] values for ATP (103 ± 12 µM), [sup 125]I-Nedd8 (0.95 ' 0.18 µM), and HsUbc12 (43 ' 13 nM) similar to those for ubiquitin activation by Uba1. Wild type [sup 125]I-ubiquitin fails to support AppBp1-Uba3 catalyzed activation or HsUbc12 transthiolation. However, modest inhibition of [sup 125]I-Nedd8 ternary complex formation by unlabeled ubiquitin suggests a K[sub d] > 300 µM for ubiquitin. Alanine 72 of Nedd8 is a critical specificity determinant for AppBp1-Uba3 binding because [sup 125]IUbR72L undergoes heterodimer-catalyzed hyperbolic HsUbc12 transthiolation and yields K[sub m] = 20 ± 9 µM and k[sub cat] = 0.9 ± 0.3 s[sup -1]. These observations demonstrate remarkable conservation in the mechanism of AppBp1Uba3 that mirrors its sequence conservation with the Uba1 ubiquitin-activating enzyme. [ABSTRACT FROM AUTHOR]