Reaction time courses were then analyzed using the kinetic simulation program KINSIM. KINSIM uses numerical integration to predict the time dependence of a reaction from a given mechanism and initial concentrations without relying upon the assumptions of steady state kinetic analysis.. G:T mismatchspecific thymine glycosylase activity of MED.Arrows mark the expected migration of the substrate and product bands.The sequence of the bottom strand of the substrate is reported on the left.The product band comigrates with the mismatched T. C, upper panel, MALDITOF mass spectrum of annealed starting G:T mismatched duplex oligonucleotide untreated with MED. The unreacted oligonucleotides served as internal standards for mass calibration.Based on the homologies with these enzymes, MED was assayed for glycosylase activity on mismatched bases.The products of the reaction were treated with strong alkali to cleave at AP sites and then were separated by electrophoresis on denaturing polyacrylamide gels.A sequencing ladder indicated that the migration of the cleavage product corresponds to the site of the mismatched thymine, or when MED was incubated with matched unmethylated, hemimethylated, or fully methylated oligonucleotide substrates, or with substrates containing extrahelical bases. These results suggest that MED has thymine glycosylase activity specific for G:T mismatches.We next conducted a MALDITOF mass spectrometry analysis of the MED glycosylase reaction with a G:T substrate.This experiment provides conclusive evidence on the thymine glycosylase activity of MED.A product band was detected for fractions after incubation with alkali; no cleavage product was detected for these fractions when this incubation was omitted.These data indicate that MED is a monofunctional glycosylase that lacks a detectable lyase activity.Mth in that all three enzymes may counteract mutagenesis caused by spontaneous deamination of methylcytosine to thymine, which would give rise to a G:T mismatch. MED was incubated with oligonucleotide substrates in which the mismatched G is immediately to A, C, G, T, or M.Mth and TDG have a mismatchspecific uracil glycosylase activity. Based on the similarities with these enzymes, we tested the uracil glycosylase activity of MED on oligonucleotide substrates in which uracil was paired with A, C, G, or T.As expected, MED uracil glycosylase activity is specific for G:U mismatches. MED efficiently removed the uracil analog FU opposite a guanine. Arrows mark the expected migration of the substrate and product bands.Arrows mark the expected migration of the substrate and product bands.MED displayed FU glycosylase activity specific for G:FU mismatches.The G:U oligonucleotide substrate constitutes a positive control.Arrows mark the expected migration of the substrate and product bands.Kinetic studies were preceded by experiments in which we assayed the glycosylase activity of MED under various reaction conditions.The reaction curve is biphasic with an initial burst followed by a slower phase. Nearmaximum product generation takes place after approximately h; for the subsequent h, there is almost no additional formation of product and the reaction proceeds at a very slow rate. This and subsequent experiments were conducted at equimolar or nearequimolar concentrations of substrate and enzyme. Under these conditions, of the substrate was typically converted into product in h.When the assay was performed with different enzyme concentrations, all lower than substrate concentration, an estimate of the active enzyme concentration could be derived.