Ub and UBL-dependent metabolism is a multi-enzyme process involving the successive activities of distinct conjugating enzymes. The E1 activating enzymes are central to ubiquitin or ubiquitin-like modifier (UBL) conjugation in vivo and in vitro since they initiate the cascade of conjugation by activating their respective modifiers. Each activating enzyme works in a parallel, but distinct way. The E1 for UBL NEDD8 will not activate SUMO, for example. Each enzyme catalyzes the reaction of ATP and Ub or UBL to activate the C-terminus of the modifier with a high energy thiolester bond. It is this high energy bond that is then transferred to the E2 carrier enzyme for the modifier for the next step in conjugation.
These enzymes are part of a larger E1-like enzyme super-family which includes roles in the biosynthesis of cysteine, thiamine, molybdenum cofactor (MoCo), and several secondary metabolites. The basic catalytic mechanism for these enzymes involved adenylation and sulfotransfer along with specific domains and structures to dictate specificity in substrate binding and other relevant protein-protein interactions. All E1 enzymes have an adenylation domain (two ThiF-homology motifs) that bind to and adenylate the Ub/UBL, a catalytic cysteine domain to which the Ub/UBL transiently attaches, and a C-terminal Ub-fold domain (UFD) which recruits E2 enzymes.
The conjugation process is initiated by the ATP-dependent formation of a thioester bond between the conserved C-terminal glycine of the Ub/UBL and the active site thiol of an E1 activating enzyme. This is an ATP-dependent process which proceeds through an E1-bound (Ub/UBL)-Gly-adenylate intermediate. This charged C-terminal residue eventually becomes conjugated to the lysyl ε-amino group of target proteins to form isopeptide linkages and subsequent conjugates. In the second step, the Ub/UBL is transferred from E1 to the active site thiol of a conjugating E2 enzyme. Finally, E3 ligases enzymes, catalyze the transfer of Ub/UBL from E2 to specific lysines of the targeted protein. E3 ligases are a large and diverse class of enzymes and are the primary determinants of substrate specificity.
The Ub E1 is highly conserved in human, yeast and plants. The primary E1 for Ub (UBE1, Uba1) contains two active sites, and activates the C-terminus of ubiquitin via a two-step intramolecular ATP-dependent reaction. Initially, a tightly enzyme-bound ubiquitin adenylate (with PPi from ATP) is formed. This intermediate is then converted to form AMP and a covalent enzyme-ubiquitin thioester. Activation of a second ubiquitin molecule gives a ternary complex with one equivalent each of ubiquitin thioester and tightly bound ubiquitin adenylate per subunit of enzyme. There exists a second, alternate E1 enzyme for Ub (UBE1L2, Uba6) which functions in conjugation with a newly identified E2 (Use1) enzyme. The UBL1L2 enzyme shares 40% identity with UBE1 and orthologs are present in vertebrates (but not in insects, worms, fungi or plants). This E1 enzyme does not activate any other UBL proteins (including SUMO, NEDD8, ISG15, FUB1, Urm1) except for FAT10, and shows comparable rates of Ub activation to UBE1 in vitro. UBE1L2 is about 10-fold less abundant than UBE1 intracellularly, but likely represents an important alternate but distinct ubiquitination pathway that is differentially regulated.
Similarly, there are cognate E1 activating enzymes for other UBLs that function in an analogous manner to UBE1. These include UBE1L (for ISG15), Uba5 (for UFM1), Uba7 (for Apg8, Apg12), SAE1/SAE2 (for SUMO), APP1/Uba3 (for NEDD8). The E1 enzymes for SUMO and NEDD8 form stable heterodimeric complexes, while all other E1 proteins are single chain enzymes.
Nedd8 - E1 Enzyme (NAE Inhibitor)
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