| Abstract
| - Covalent conjugation of ubiquitin to intracellular proteins is asignal for degradation by the26S protease. Conjugation is usually accomplished by thesequential action of activating (E1), conjugating(E2), and ligase (E3) enzymes. Each of these enzymes forms acovalent thiol ester with ubiquitin as partof its catalytic cycle. In most cases, the apparent role of theubiquitin conjugating enzyme (E2) is totransfer ubiquitin from the E1 active site to the E3 active site.Ubiquitin is then delivered from E3 to thesubstrate lysine residue. An unusually large,reticulocyte-specific enzyme, known as E2-230K, is uniqueamong the large family of E2 enzymes is being susceptible to inhibitionby inorganic arsenite [Klempereret al. (1989) Biochemistry 28, 6035−6041]. We showthat phenylarsenoxides potently inhibit E2-230K,apparently by binding to vicinal Cys residues of the enzyme: boundaminophenylarsenoxide partiallyprotects the enzyme against inactivation by N-ethylmaleimide(NEM), and prior enzyme inactivation withNEM blocks enzyme binding to immobilized phenylarsenoxide. Studieson the mechanistic basis ofinhibition showed that a concentration of (aminophenyl)arsenoxidethat produced complete inhibition ofsteady-state turnover had no effect on the turnover of the preformedE2−ubiquitin adduct. However,when the enzyme was preincubated with this concentration of inhibitorprior to initiation of adductformation, the level of E2-associated ubiquitin was reduced by 60%.These results are consistent with amodel in which two Cys residues of the enzyme sequentially form thiolesters with ubiquitin and thesecond of these Cys residues is bound to arsenic in theenzyme-inhibitor complex. In this model, E2-230K functions as an E2−E3 hybrid.
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