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. 2013 Jun 17;4:1982. doi: 10.1038/ncomms2982

Figure 3. Catalytic machinery of Parkin.

Figure 3

(a) The activity-probe HA-Ub-VS was incubated with various Parkin constructs (or USP2 control) to determine intrinsic Parkin enzymatic activity. Reactions were allowed to proceed for 3 h, or for the Parkin blot (loading control) samples were removed at time zero (t0) hours and terminated by the addition of SDS-loading dye. For RBR samples, the reaction proceeds so quickly that even at time zero, the reaction is observed. (b) The potential catalytic triad residues C431, H433 and E444 are misaligned. H433 is engaged in a water-mediated hydrogen bond with W462 and is ~5.1 Å from C431. A GG-C431 motif is present (asterisks), which could serve as a classical oxyanion hole during catalysis. (c) Parkin probe reactivity requires elements of a classical catalytic triad. Ub-VS probe reactivity was assessed in various Parkin mutants over a range of pH.