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 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 microm), 125I-Nedd8 (0.95 +/- 0.18 microm), 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 microm 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 microm 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.
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Richard N
RN
Bohnsack
Arthur L
AL
Haas
D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004912:Erythrocytes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D000075687:NEDD8 Protein; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes; D014452:Ubiquitins / Q000737:chemistry* / Q000378:metabolism*
D016564:Amyloid beta-Protein Precursor; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D011994:Recombinant Proteins; D014452:Ubiquitins; D044764:Ubiquitin-Activating Enzymes; C491802:NAE protein, human