Hydrolysis Kinetics

Enzymatic hydrolysis of dinucleotide cap analogs was performed using DcpS at a final concentration of 30 nM (in experiments with m⁷GpppG) and at a final concentration of 5 nM (in experiments with m₃^2,2,7GpppG). The substrate concentration in the reaction mixture was 10 μM. The reactions were conducted at 20 °C in 50 mM Tris–HCl buffer containing 150 mM NaCl, pH 7.2. The fluorometric method, where the increase in fluorescence due to DcpS-mediated cap dinucleotide hydrolysis is recorded, was used to measure the progress of cap analog hydrolysis.²² The reactions were monitored for 30–60 min by recording the time-dependent increase of the fluorescence intensity, which is caused by removal of intramolecular stacking as a result of enzymatic cleavage of the dinucleotide cap analogs. On the basis of fluorometric data, the fraction of the generated product was plotted against time, and the pseudo-first order enzymatic decapping rate constants were calculated.⁴⁰ Fluorescence experiments were performed using a LS-50B spectrofluorometer (Perkin-Elmer Co., Norwalk, CT, USA).