Trivalent Arsenicals: Probes of Mechanism and Specificity inReactions of Ubiquitin-Protein Ligation

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Trivalent arsenical reagents interact specificially with proteins bearing vicinal sulfhydryl groups, forming very stable cyclic dithioarsenite complexes. Such complexes are inactive if the sulfhydryl groups are critical catalytic or regulatory elements. Ubiquitin (Ub) is a small and highly-conserved polypeptide whose ligation to other cellular proteins can bring about protein degradation. In some cases, Ub ligation may also serve to regulate protein function. Trivalent arsenicals have been found to inhibit two enzymes involved in Ub protein ligation. One of these, a Ub carrier protein (E2-230K), directly catalyzes Ub conjugation to proteins. The other is an aminoacyl-tRNA protein transferase. Amino-terminal arginylation catalyzed by the transferase renders proteins substrates for Ub ligation. The proposed research addresses the mechanistic and structural implications of inhibition of these enzymes by trivalent arsenicals. The experiments will reveal whether inhibition is indeed mediated by interaction of arsenicals with vicinal sulfhydryls, and will also address how this structural element, if present, functions in catalysis and/or regulation. This research addresses basic issues concerning catalytic mechanism and specificity in Ub-protein ligation, and may also lead to development of better and more specific inhibitors.