title: Calorimetric studies of ligand binding in R67 dihydrofolate reductase.
abstract: R67 dihydrofolate reductase (DHFR) is a novel bacterial protein that possesses 222 symmetry and a single active site pore. Although the 222 symmetry implies that four symmetry-related binding sites must exist for each substrate as well as for each cofactor, various studies indicate only two molecules bind. Three possible combinations include two dihydrofolate molecules, two NADPH molecules, or one substrate plus one cofactor. The latter is the productive ternary complex. To explore the role of various ligand substituents during binding, numerous analogues, inhibitors, and fragments of NADPH and/or folate were used in both isothermal titration calorimetry (ITC) and K(i) studies. Not surprisingly, as the length of the molecule is shortened, affinity is lost, indicating that ligand connectivity is important in binding. The observed enthalpy change in ITC measurements arises from all components involved in the binding process, including proton uptake. As a buffer dependence for binding of folate was observed, this likely correlates with perturbation of the bound N3 pK(a), such that a neutral pteridine ring is preferred for pairwise interaction with the protein. Of interest, there is no enthalpic signal for binding of folate fragments such as dihydrobiopterin where the p-aminobenzoylglutamate tail has been removed, pointing to the tail as providing most of the enthalpic signal. For binding of NADPH and its analogues, the nicotinamide carboxamide is quite important. Differences between binary (binding of two identical ligands) and ternary complex formation are observed, indicating interligand pairing preferences. For example, while aminopterin and methotrexate both form binary complexes, albeit weakly, neither readily forms ternary complexes with the cofactor. These observations suggest a role for the O4 atom of folate in a pairing preference with NADPH, which ultimately facilitates catalysis.
authors.affiliation: Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Jackson
authors.forename: Shaileja
authors.initials: S
authors.lastname: Chopra
authors.forename: R Derike
authors.initials: RD
authors.lastname: Smiley
authors.forename: Patrick O'Neal
authors.initials: PO
authors.lastname: Maynord
authors.forename: Andre
authors.initials: A
authors.lastname: Rosowsky
authors.forename: Robert E
authors.initials: RE
authors.lastname: London
authors.forename: Louis
authors.initials: L
authors.lastname: Levy
authors.forename: Thomas I
authors.initials: TI
authors.lastname: Kalman
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Howell
mesh_terms: D002151:Calorimetry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005492:Folic Acid / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D005493:Folic Acid Antagonists; D008024:Ligands; D011994:Recombinant Proteins; D009249:NADP; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase