title: Thermodynamic and NMR analysis of inhibitor binding to dihydrofolate reductase.
abstract: Isothermal titration calorimetry (ITC) was used to determine the thermodynamic driving force for inhibitor binding to the enzyme dihydrofolate reductase (DHFR) from Escherichia coli. 1,4-Bis-{[N-(1-imino-1-guanidino-methyl)]sulfanylmethyl}-3,6-dimethyl-benzene (1) binds DHFR:NADPH with a K(d) of 13±5 nM while the related inhibitor 1-{[N-(1-imino-guanidino-methyl)]sulfanylmethyl}-3-trifluoromethyl-benzene (2) binds DHFR:NADPH with a K(d) of 3.2±2.2 μM. The binding of these inhibitors has both a favorable entropy and enthalpy of binding. Additionally, we observe positive binding cooperativity between both 1 and 2 and the cofactor NADPH. Binding of compound 1 to DHFR is 285-fold tighter in the presence of the NADPH cofactor than in its absence. We did not detect binding of 2 to DHFR in the absence of NADPH. The backbone amide (1)H and (15)N NMR resonances of DHFR:NADPH and both DHFR:NADPH inhibitor complexes were assigned in order to better understand the binding of these inhibitors in solution. The chemical shift perturbations observed with the binding of 1 were greatest at residues closest to the binding site, but significant perturbations also occur away from the inhibitor location at amino acids in the vicinity of residue 58 and in the GH loop. The pattern of chemical shift changes observed with the binding of 2 is similar to that seen with 1. The main differences in chemical shift perturbation between the two inhibitors are in the Met20 loop and in residues at the interface between the inhibitor and NADPH.
authors.affiliation: Department of Chemistry, York University, 4700 Keele St., Toronto, Ontario, Canada M3J 1P3.
authors.forename: Ihor
authors.initials: I
authors.lastname: Batruch
authors.forename: Eliott
authors.initials: E
authors.lastname: Javasky
authors.forename: Eric D
authors.initials: ED
authors.lastname: Brown
authors.forename: Michael G
authors.initials: MG
authors.lastname: Organ
authors.forename: Philip E
authors.initials: PE
authors.lastname: Johnson
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001555:Benzene Derivatives; D001665:Binding Sites / Q000187:drug effects; D002151:Calorimetry; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005493:Folic Acid Antagonists / Q000737:chemistry*; D006146:Guanidines; D009682:Magnetic Resonance Spectroscopy; D009249:NADP / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000187:drug effects / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D000900:Anti-Bacterial Agents; D001555:Benzene Derivatives; D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D006146:Guanidines; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase