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-H-3] ATP and I-125-Nedd8, heterodimer rapidly forms a stable stoichiometric ternary complex composed of tightly bound Nedd8 [H-3] adenylate and Uba3-I-125-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 I-125-Nedd8 (k(cat) = 3.5 +/- 0.2 s(-1)), yielding K-m values for ATP (103 +/- 12 muM), I-125-Nedd8 (0.95 +/- 0.18 muM), and HsUbc12 (43 +/- 13 nM) similar to those for ubiquitin activation by Uba1. Wild type I-125-ubiquitin fails to support AppBp1-Uba3 catalyzed activation or HsUbc12 transthiolation. However, modest inhibition of I-125-Nedd8 ternary complex formation by unlabeled ubiquitin suggests a K-d > 300 muM for ubiquitin. Alanine 72 of Nedd8 is a critical specificity determinant for AppBp1-Uba3 binding because I-125-UbR72L undergoes heterodimer-catalyzed hyperbolic HsUbc12 transthiolation and yields K-m = 20 +/- 9 muM 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.