Effects of Molecular Crowding on the Binding Affinity of Dihydrofolate for Dihydrofolate Reductase

Abstract

The reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) by the enzyme dihydrofolate reductase (DHFR), using NADPH as a cofactor, is an essential part of the folate cycle. The inhibition of DHFR leads to interruption of DNA synthesis and consequently cell death, making this enzyme a crucial target in the treatment of cancer and other diseases. Previous studies examined the effects of small molecule osmolytes on the substrate interactions with two non-homologous DHFRs, E. coli chromosomal DHFR (EcDHFR) and R67 DHFR, with vastly different active site structures. The results indicated that DHF weakly interacts with the osmolytes in solution, shifting the binding equilibrium from DHF bound to DHFR to unbound DHF. It is hypothesized that similar weak, nonspecific interactions may also occur between cellular proteins and DHF. Weak interactions between cellular proteins and DHF would have consequences in vivo, where the concentration of the cellular milieu is approximately 300 g/L. Under the crowded conditions in the cell, there is a higher propensity for intermolecular interaction.Crowding effects of macromolecules in concentrations similar to those in vivo were examined. Isothermal titration calorimetry (ITC) and enzyme kinetic assays were used to detect effects of molecular crowders by monitoring activity of the (DHFR)-NADPH or DHF complex and the ternary DHFR-DHF-NADPH complex in the presence of these crowders. To recreate the conditions of molecular crowding in vivo, the binding of the enzyme-ligand complexes in the presence of molecular weight crowding agents (lysozyme or casein) was examined. Analysis of the Kd's and Km's indicated a correlation between increased molecular crowding in the solution and weakened binding of the DHFR-substrate complexes. These findings indicate an importance of molecular crowding on EcDHFR activity in vivo.