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Figure 1: Thioester Activity Assay: UBE2D1 (UbcH5a) forms a thioester with Ub in an ATP-dependent manner and the bond can be reduced with addition of access DTT. Confirms the activity of UBE2D1.
UBE2D1 (UbcH5a) is an E2 ubiquitin conjugating enzyme. An E1 activating enzyme is required to attach ubiquitin to UBE2D1 via an active site cysteine. The mechanism of ubiquitin transfer involves the breaking of a E1-Ub thioester linkage, followed by a reformation of a UBE2D1-Ub thioester. UBE2D1 is capable of associating with numerous known E3 ligases which target abnormal proteins for proteasomal degradation through polyubiquitination. UBE2D1 is also known to interact with Parkin and to be involved in PINK1 mediated mitophagy.