- X-ray Crystallography: This technique can reveal the three-dimensional structure of the enzyme, including secondary binding sites. - NMR Spectroscopy: NMR spectroscopy can provide information on the dynamics and interactions at secondary binding sites. - Mutagenesis Studies: Introducing mutations at or near the secondary site can help understand its role in catalysis. - Computational Modeling: Molecular dynamics simulations and other computational methods can predict the effects of secondary site interactions on enzyme function.
