MECHANISM OF UBIQUITIN-CONJUGATING ENZYME E2-230K - CATALYSIS INVOLVING A THIOL RELAY

Covalent conjugation of ubiquitin to intracellular proteins is a signa l for degradation by the 26S protease. Conjugation is usually accompli shed by the sequential action of activating (El), conjugating (E2), an d ligase (E3) enzymes. Each of these enzymes forms a covalent thiol es ter 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 El active site to the E3 active site. Ubiquitin is then delivered from E3 to the substrate lysine residue. An unusually l arge, reticulocyte-specific enzyme, known as E2-230K, is unique among the large family of E2 enzymes is being susceptible to inhibition by i norganic 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 aminophenylars enoxide partially protects the enzyme against inactivation by N-ethylm aleimide (NEM), and prior enzyme inactivation with NEM blocks enzyme b inding to immobilized phenylarsenoxide. Studies on the mechanistic bas is of inhibition showed that a concentration of (aminophenyl)arsenoxid e that produced complete inhibition of steady-state turnover had no ef fect on the turnover of the preformed E2-ubiquitin adduct. However, wh en the enzyme was preincubated with this concentration of inhibitor pr ior to initiation of adduct formation, the level of E2-associated ubiq uitin was reduced by 60%. These results are consistent with a model in which two Cys residues of the enzyme sequentially form thiol esters w ith 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.