Direct measurement of drug–enzyme interactions by atomic force microscopy; dihydrofolate reductase and methotrexate

Abstract

There is a wealth of structural information on the drug–enzyme complex, dihydrofolate reductase and methotrexate. However, the dissociation dynamics of the complex remain relatively poorly understood. This paper describes the application of the atomic force microscope to quantify the rupture forces upon the forced dissociation of the ligand from the enzyme. The intermolecular forces experienced between dihydrofolate reductase and methotrexate as a binary and ternary complex, and the effect of pH upon these forces were studied. The rate at which forced dissociation occurs is known to have a significant effect upon the forces experienced between receptor–ligand complexes. This dependency of rupture force upon retract velocity is investigated. A linear relationship between force and the logarithm of velocity is demonstrated for the forced dissociation of the binary complex, inferring the presence of a barrier in the energy landscape positioned approximately 3 Å away from the bound state. The influence of the cofactor, NADPH, upon these rupture forces was negligible, suggesting that the barrier probed does not facilitate the cooperativity of the ternary complex. However, protonation of the Asp26 residue situated deep within the methotrexate binding site results in a decrease in rupture force.