Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2).Mechanistic studies on Compound I and its purified ubiquitin adduct demonstrate that the proposed substrate-assisted inhibition via covalent adduct formation is entirely consistent with the three-step ubiquitin activation process and that the adduct is formed via nucleophilic attack of UAE thioester by the sulfamate group of Compound I after completion of step 2. Kinetic and affinity analysis of Compound I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of adduct formation and the affinity between the adduct and E1 contribute to the overall potency. Because all E1s are thought to use a similar mechanism to activate their cognate ubiquitin-like proteins, the substrate-assisted inhibition by adenosine sulfamate analogues represents a promising strategy to develop potent and selective E1 inhibitors that can modulate diverse biological pathways.Post-translational modification by ubiquitin plays an essential role in a wide range of cellular processes. One of the most intensively studied pathways is the ubiquitin-proteasome system that attaches a polyubiquitin chain to a lysine residue on a target protein and directs it to proteasome-mediated proteolysis. The ubiquitin-proteasome system is a key system responsible for maintaining cellular protein homeostasis, an emerging research area that could potentially transform our understanding of human diseases (1-3). Bortezomib (VELCADE), a proteasome inhibitor, is currently approved in the treatment of patients with multiple myeloma and relapsed mantle cell lymphoma (4, 5). The clinical success of bortezomib suggests that targeting other components in the ubiquitin-proteasome system pathway might represent an opportunity to develop novel anti-cancer therapeutics (6 -9).Conjugating ubiquitin to a protein substrate is mediated by an enzymatic cascade initiated by ubiquitin-activating enzyme (UAE) 2 (or Ube1 in humans, also known as E1) (10). Previous mechanistic studies show that, in vitro, UAE activates ubiquitin by a three-step process using ATP as a cofactor (Fig. 1A) (11,12). In step 1, UAE binds ATP and ubiquitin, catalyzes formation of ubiquitin adenylate intermediate, and releases inorganic pyrophosphate (PP i ). The ubiquitin adenylate activates the C-terminal carboxyl group of ubiquitin for nucleophilic substitution. In step 2, the catalytic cysteine residue in UAE attacks the adenylate to form a thioester intermediate (UAE-Sϳubiq-uitin, ϳ denotes the thioester bond between the C-terminal carboxyl group of ubiquitin and Cys 632 of human UAE) with AMP as the by-product. In step 3, UAE-Sϳubiquitin binds another equivalent of ATP and ubiquitin and in a second round of adenylation, forms a UAE-Sϳubiquitin⅐ubiquitin-adenylate ternary complex. Although UAE-Sϳubiquitin is capable of transferring ubiquitin to the conjugating enzyme (E2) via a transthiolation reaction, E1-E2 transthiolation is grea...

Background:The Uba6 pathway and its components play an important role in a variety of biological processes. Results: The mechanism of how Uba6 activates two distinct substrates, ubiquitin and FAT10, was characterized. Conclusion: Uba6 was shown to use a similar mechanism for activating both substrates with a greater affinity for FAT10. Significance: Relative levels of ubiquitin and FAT10 could regulate the Uba6 pathway in cells.

Background: Human ubiquitin-like protein Ufm1 and its activating enzyme, Uba5, are involved in endoplasmic reticulum (ER) stress response. Results: The Uba5-Ufm1 conjugation pathway is characterized in both reconstituted systems and cellular settings. Conclusion: Uba5 activates Ufm1 via a distinct two-step mechanism. Significance: This study represents the first mechanistic characterization of Uba5, a homodimeric member of the E1 enzyme family.

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