Fig. 3: ATP hydrolysis of Dop is allosterically influenced by the Dop-loop. | Nature Communications

Fig. 3: ATP hydrolysis of Dop is allosterically influenced by the Dop-loop.

From: Structures of prokaryotic ubiquitin-like protein Pup in complex with depupylase Dop reveal the mechanism of catalytic phosphate formation

Fig. 3: ATP hydrolysis of Dop is allosterically influenced by the Dop-loop.

a The Dop-loop variant (DopGS) exhibits a longer phase in the depupylation time course of the fluorescent model substrate Pup-(5-FAM-Lys) (Pup-Fl) than the wild type (WT) enzyme in the presence of ATP. 1.25 µM CgluDop (WT or DopGS variant) was incubated with 5 μM Pup-Fl and 100 µM ATP or ADP plus 10 mM Na/K Pi (pH 8.0 at 23 °C) at 30 °C. The measurement was started by addition of nucleotide. Each line represents the average of three independent replicates. b Steady-state enzyme kinetic analysis of depupylation of Pup-Fl by using fluorescence anisotropy. Each experiment was carried out in three independent replicates and data are represented as mean values ± SD. c Depupylation time courses of PanB-Pup catalyzed by Dop variants in the presence of ATP. PanB-Pup (3 µM) was incubated with 0.5 µM CgluDop (DopWT or DopGS) at 30 °C supplemented with 0.5 mM ATP. d Densitometric analysis of PanB bands in (c) with respect to the total amount of PanB-Pup used in the reaction. Each reaction was carried out in two or three independent replicates and data are represented as mean values ± SD.

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