Fig. 2.

Overview of KIE measurement under competitive conditions. (A) Schematic description of a competition reaction with a pair of isotopic cofactors and the resultant overlapping MS data with CH₃/CD₃ and CH₃/¹³CD₃ cofactor pairs. The reaction sample was split into two portions for partial consumption and full consumption of the pair of isotopic cofactors, respectively. Their light-to-heavy isotope ratios (blue vs. red) are quantified by MS as described in Materials and Methods. (B) Schematic description of BIE measurement under competitive conditions. Here a pair of prebound or SET8-bound CH₃/CD₃ isotopic cofactors were converted to label H4K20 peptide substrate under competitive conditions. The resultant methylated H4K20 peptide was subjected to MS analysis to quantify the isotopic ratios as described in Materials and Methods.