Binding versus Enzymatic Processing of ε-Trimethyllysine Dioxygenase Substrate Analogues

epsilon-Trimethyllysine dioxygenase (TMLD) is a nonheme Fe(II) and alpha-ketoglutarate dependent oxygenase that catalyzes the stereospecific hydroxylation of epsilon-trimethyl-L-lysine (TML) to beta-hydroxy-TML during the first step of L-carnitine biosynthesis. Targeting TMLD with inhibitors is a viable strategy for the treatment of cardiovascular diseases. Herein, we report a methodology for isothermal titration calorimetry analysis of TMLD substrate analogue binding to the enzyme. Despite the high structural similarity of the tested compounds, two different binding mechanisms (enthalpy- and entropy-driven) were observed, giving insight into the ligand (substrate) selectivity of TMLD. We demonstrate that the method allows distinguishing a natural substrate-like binding mode, which correlates with the ability of the compounds to serve as substrates in the TMLD catalytic reaction.