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This indicates that each molecule of active MED enzyme efficiently removes only approximately one mismatched thymine.To further explore this nearstoichiometric relationship and rule out the artifactual possibility that a <a href="http://inhibit09.online/archives/223"></a> fraction of substrate could not be converted into product, we added additional aliquots of enzyme after the reaction reached its slow phase.This resulted in additional conversion of substrate into product, showing that the remaining substrate was competent to partake in   the reaction.In order to discriminate between the two possibilities, we conducted an analysis of the kinetic data.Because the kinetic studies were conducted under conditions of similar concentrations of substrate and enzyme, and within a h incubation, the assumptions required for steady state kinetic analysis are not applicable.In particular, in our experiments, the concentration of enzyme is not much smaller than the concentrations of substrateproduct, and there is only a slow enzyme turnover within the reaction time course.Applying standard steady state kinetic analyses, under the required experimental conditions of enzyme concentration much less than substrate concentration, would thus require impractically long reaction times. We therefore analyzed the data using the kinetic simulation program KINSIM.KINSIM utilizes numerical integration to predict the time course of a reaction as governed by a kinetic mechanism and the reactant concentrations. Arrows mark the expected migration of the substrate and product bands.C, nearstoichiometric relationship between MED enzyme and substrateproduct.The preincubation was followed by incubation with a radioactive G:T substrate.Thus, this confirms that the biphasic kinetics of MED glycosylase reaction is a consequence of high product affinity with slow release rather than enzyme inactivation.Assuming that the koff of the AP site is the same as determined for the G:T substrate and that the rate of release of the free uracil is similar to that of free thymine, the KINSIM simulation should fit the kinetic data simply by changing the chemical step.Indeed, the kcat is the major difference between the G:T and G:U substrate: uracil is removed by MED from G:U mismatches faster than thymine is removed from G:T mismatches.In order to test the alternate possibility that the differences in glycosylase activity result from different binding of MED to the two DNA substrates, we conducted kinetic analysis at increasing equimolar concentrations of MED and G:T or G:U substrates, and nM concentrations, indicating that at these concentrations binding of the enzyme to the two substrates is essentially saturated. This also confirms the KINSIM prediction that the different reaction rate on G:U and G:T substrates is not due to differences in substrate binding but rather reflects intrinsic differences in kcat. To test this possibility, we compared the thymine glycosylase activity of wild type MED and a recombinant deletion mutant lacking the MBD and encompassing only the catalytic domain. Thus, the catalytic domain of MED is necessary and sufficient for glycosylase activity, whereas the MBD is dispensable in this assay.Thus, methylation of the mismatched substrate is not required for efficient catalysis by MED.The uracil glycosylase activity of MED is limited to G:U mismatches; MED does not have glycosylase activity on uracil paired with adenine, cytosine, or thymine in doublestranded DNA substrates, or on uracil present in singlestrand DNA.

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