Fluorescent Analysis of KM and Vmax Values for Methyl-Dependent Restriction Endonucleases

Methyl-dependent restriction endonucleases are promising tools for analyzing eukaryotic DNA methylation patterns. However, quantitative assessment of their substrate specificity requires the determination of the kinetic parameters of enzymatic reactions. Here, we present a method for determining initial reaction rates based on fluorescent probes and real-time monitoring of changes in fluorescence intensity. Initial rates of methyl-dependent GlaI and BlsI restriction endonucleases were determined as the slope of the linear part of the kinetic curves, after which the Michaelis–Menten constants (KM) and reaction rates (Vmax) were calculated using nonlinear regression. For both enzymes, KM values were determined for the first time, indicating a high affinity of the methyl-dependent restriction endonucleases for methylated sites. KM values for fully methylated duplexes were in the range of (4.4–7.0)·102 nM for GlaI and 2.4–55 nM for BlsI. KM values were significantly lower for the hemimethylated duplexes: (1.9–8.0)·102 nM for GlaI and (0.7–15.0)·102 nM for BlsI; and even lower for unmethylated duplexes: (27–46)·102 nM for GlaI and (0.28–23)·102 nM for BlsI. Maximum reaction rates varied within relatively narrow limits: Vmax values were in range (4.5–19.5)·10−4 nM/s for GlaI and (1.4–18)·10−4 nM/s for BlsI, respectively. Vmax values were depended weakly on the degree of methylation compared to the KM. The proposed fluorescence method was applied to determine the kinetic parameters of methyl-dependent restriction endonucleases for the first time. It may serve as a simpler and more environmentally friendly alternative to traditional electrophoretic approaches that use radioactive labels.