Thermodynamic Studies of Myristoyl-CoA: Protein N-Myristoyltransferase Using Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) is a technique used widely to examine various aspects of enzyme function and regulation including kinetic reaction mechanisms, the chemical basis of catalysis, and determinants of substrate and inhibitor binding specificity. The utility of the ITC method is illustrated by an analysis of the effects of varying acyl chain length on the energetics of interaction between myristoyl-CoA: protein N-myristoyltransferase (NMT) and acyl-CoA ligands and on subsequent interactions of NMT: acyl-CoA binary complexes with peptide substrates. NMT has a highly ordered kinetic mechanism, that cooperative interactions exist between the myristoyl- CoA and peptide binding sites of the enzyme, and that the energy of binding C16 acyl-CoAs is sufficient to induce the cooperative transition which permits binding of peptide but not to generate a fully functional active site. ITC measures the heat evolved during molecular associations. It allows determination of the standard Gibbs free energy change (ΔG°), the enthalpy change (ΔH°), the entropy change (ΔS°), and the stoichiometry of the binding event. Determining the binding constant for a molecular association requires measurement of the free and bound concentrations of the interacting molecules.