Fluorescent Analysis of K_M and V_max 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 (K_M) and reaction rates (V_max) were calculated using nonlinear regression. For both enzymes, K_M values were determined for the first time, indicating a high affinity of the methyl-dependent restriction endonucleases for methylated sites. K_M 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. K_M 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: V_max 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. V_max values were depended weakly on the degree of methylation compared to the K_M. 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.