title: Thermodynamics and solvent effects on substrate and cofactor binding in Escherichia coli chromosomal dihydrofolate reductase.
abstract: Chromosomal dihydrofolate reductase from Escherichia coli catalyzes the reduction of dihydrofolate to tetrahydrofolate using NADPH as a cofactor. The thermodynamics of ligand binding were examined using an isothermal titration calorimetry approach. Using buffers with different heats of ionization, zero to a small, fractional proton release was observed for dihydrofolate binding, while a proton was released upon NADP(+) binding. The role of water in binding was additionally monitored using a number of different osmolytes. Binding of NADP(+) is accompanied by the net release of ∼5-24 water molecules, with a dependence on the identity of the osmolyte. In contrast, binding of dihydrofolate is weakened in the presence of osmolytes, consistent with "water uptake". Different effects are observed depending on the identity of the osmolyte. The net uptake of water upon dihydrofolate binding was previously observed in the nonhomologous R67-encoded dihydrofolate reductase (dfrB or type II enzyme) [Chopra, S., et al. (2008) J. Biol. Chem. 283, 4690-4698]. As R67 dihydrofolate reductase possesses a nonhomologous sequence and forms a tetrameric structure with a single active site pore, the observation of weaker DHF binding in the presence of osmolytes in both enzymes implicates cosolvent effects on free dihydrofolate. Consistent with this analysis, stopped flow experiments find betaine mostly affects DHF binding via changes in k(on), while betaine mostly affects NADPH binding via changes in k(off). Finally, nonadditive enthalpy terms when binary and ternary cofactor binding events are compared suggest the presence of long-lived conformational transitions that are not included in a simple thermodynamic cycle.
authors.affiliation: Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, USA.
authors.forename: Jordan
authors.initials: J
authors.lastname: Grubbs
authors.forename: Sharghi
authors.initials: S
authors.lastname: Rahmanian
authors.forename: Alexa
authors.initials: A
authors.lastname: DeLuca
authors.forename: Chetan
authors.initials: C
authors.lastname: Padmashali
authors.forename: Michael
authors.initials: M
authors.lastname: Jackson
authors.forename: Michael R
authors.initials: MR
authors.lastname: Duff
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Howell
mesh_terms: D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D002875:Chromosomes / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D009249:NADP / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
chemical_list: D008024:Ligands; D012997:Solvents; D014867:Water; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase