Thermodynamic analysis of l-arginine and Nω-hydroxy-l-arginine binding to nitric oxide synthase

Isothermal titration calorimetry has been used to determine thermodynamic parameters of substrate binding to the oxygenase domain of neuronal nitric oxide synthase (nNOSoxy) in the presence of the cofactor tetrahydrobiopterin. The intermediate Nω-hydroxy-l-arginine (NHA) has a larger affinity than l-Arginine (l-Arg) for nNOSoxy, with Kd = 0.4 ± 0.1 µM and 1.7 ± 0.3 µM at 25 °C, respectively. nNOSoxy binds NHA and l-Arg with ΔH − 4.1 ± 0.2 and − 1.0 ± 0.1 kcal/mol and ΔS = 15 and 23 cal/Kmol respectively. NHA binding is more exothermic probably due to formation of an extra hydrogen bond in the active site compared to l-Arg. The changes in heat capacity (ΔCp) are relatively small for binding of both NHA and l-Arg (− 53 ± 18 and − 95 ± 23 cal/L mol, respectively), which indicates that hydrophobic interactions contribute little to binding.