Mechanism of Ubiquitin Conjugating Enzyme E2-230K:  Catalysis Involving a Thiol Relay?

Abstract

Covalent conjugation of ubiquitin to intracellular proteins is a signal for degradation by the 26S protease. Conjugation is usually accomplished by the sequential action of activating (E1), conjugating (E2), and ligase (E3) enzymes. Each of these enzymes forms a covalent thiol ester with ubiquitin as part of its catalytic cycle. In most cases, the apparent role of the ubiquitin conjugating enzyme (E2) is to transfer ubiquitin from the E1 active site to the E3 active site. Ubiquitin is then delivered from E3 to the substrate lysine residue. An unusually large, reticulocyte-specific enzyme, known as E2-230K, is unique among the large family of E2 enzymes is being susceptible to inhibition by inorganic arsenite [Klemperer et al. (1989) Biochemistry 28, 6035-6041]. We show that phenylarsenoxides potently inhibit E2-230K, apparently by binding to vicinal Cys residues of the enzyme: bound aminophenylarsenoxide partially protects the enzyme against inactivation by N-ethylmalemide (NEM), and prior enzyme inactivation with NEM blocks enzyme binding to immobilized phenylarsenoxide. Studies on the mechanistic basis of inhibition showed that a concentration of (aminophenyl)arsenoxide that produced complete inhibition of steady-state turnover had no effect on the turnover of the preformed E2-ubiquitin adduct. However, when the enzyme was preincubated with this concentration of inhibitor prior to initiation of adduct formation, the level of E2-associated ubiquitin was reduced by 60%. These results are consistent with a model in which two Cys residues of the enzyme sequentially form thiol esters with ubiquitin and the second of these Cys residues is bound to arsenic in the enzyme-inhibitor complex. In this model, E2-230K functions as an E2-E3 hybrid.

Targeted Degradation of the AML1/MDS1/EVI1 Oncoprotein by Arsenic Trioxide

“…Our data show that ATO targets EVI1 for degradation, at least in part, through the ubiquitin-proteasome pathway. Arsenite has been shown to regulate ubiquitination both positively and negatively (44)(45)(46)(47)(48)(49). One interesting finding is that arsenite induces Cdc25C degradation through the KEN-box and ubiquitin-proteasome pathway (50).…”

Investigations of methylmercury-induced alterations in neurogenesis.

“…i (Berleth and Pickart 1996;Hochstrasser 1996), both of which are sensitive to MeHg exposure (Atchison and Hare 1994;Sarafian and Verity 1991). Regulated protein degradation also involves protein phosphorylationmediated cell signaling, and MeHg alters intracellular protein phosphorylation.…”

E2/E3-mediated Assembly of Lysine 29-linked Polyubiquitin Chains

“…If the E3 is less abundant, as we think likely, k cat would be larger. Reported k cat values for E2s in E3-independent conjugation range from 0.02 to 0.6 min Ϫ1 (27,33,47), whereas k cat ϳ0.5 min Ϫ1 has been reported for the E2-E3 hybrid protein E2-230K (48). No k cat value has been reported for an E3.…”