Figure 6.
NExo is an efficient 3′-phosphatase. (A) 50-PO4 (Table 1) was used as the substrate to measure removal of a 3′-blocking PO4 from an internal AP site. NExo (1 nM) was incubated with 50-PO4 (50 nM) under standard phosphatase assay conditions and aliquots removed at the indicated time points, quenched and resolved by denaturing 20% PAGE. (B) A number of reactions were performed with varying substrate concentrations using substrate 20-PO4 (Table 1); quantified data are shown with the best fit to the Michaelis–Menten equation with kcat = 0.49 ± 0.02/s and Km = 28 ± 6 nM.