Phospho-dependent Regulation of SAMHD1 Oligomerisation Couples Catalysis and Restriction
Fig 7
The effect of phosphorylation on SAMHD1 enzyme activity.
(A) Steady-state kinetic analysis of GTP stimulated hydrolysis of TTP, by SAMHD1 (115–626) (black) and phospho-SAMHD1(115–626) (blue). The dependence of the rate of on substrate concentration is plotted. Error bars represent SEM of three independent measurements. Solid lines are the best fit to the data using the Michaelis-Menten expression. The derived constants KM and kcat are shown inset. (B and C) Effect of activator depletion on SAMHD1 and pSAMHD1 triphosphohydrolase activity. (B) IEX-HPLC analysis of nucleotide composition after incubation of SAMHD1(115–626) with GTP (upper), and pSAMHD1(115–626) (middle) or SAMHD1(115–626) (lower) with dATP and GTP for 0 and 300 seconds. (C) IEX-HPLC analysis of the hydrolysis of ddGTP added to the samples depleted of dATP in (B). The plot shows the quantification of the time dependent hydrolysis derived from integration of the ddGTP peak at each time point. Error bars represent the SEM from 3 independent experiments.