title: Widespread Perturbation of Function, Structure, and Dynamics by a Conservative Single-Atom Substitution in Thymidylate Synthase.
abstract: Thymidylate synthase (TSase) is responsible for synthesizing the sole de novo source of dTMP in all organisms. TSase is a drug target, and as such, it has been well studied in terms of both structure and reaction mechanism. Cysteine 146 in Escherichia coli TSase is universally conserved because it serves as the nucleophile in the enzyme mechanism. Here we use the C146S mutation to probe the role of the sulfur atom in early events in the catalytic cycle beyond serving as the nucleophile. Surprisingly, the single-atom substitution severely decreases substrate binding affinity, and the unfavorable ΔΔG°bindis comprised of roughly equal enthalpic and entropic components at 25 °C. Chemical shifts in the free and dUMP-bound states show the mutation causes perturbations throughout TSase, including regions important for complex stability, in agreement with a less favorable enthalpy change. We measured the nuclear magnetic resonance methyl symmetry axis order parameter (S2axis), a proxy for conformational entropy, for TSase at all vertices of the dUMP binding/C146S mutation thermodynamic cycle and found that the calculated TΔΔS°confis similar in sign and magnitude to the calorimetric TΔΔS°. Further, we ascribed minor resonances in wild-type-dUMP spectra to a state with a covalent bond between Sγ of C146 and C6 of dUMP and find S2axisvalues are unaffected by covalent bond formation, indicating this reaction step is neutral with respect to ΔS°conf. Lastly, the C146S mutation allowed us to measure cofactor analog binding by isothermal titration calorimetry without the confounding heat signature of covalent bond formation. Raltitrexed binds free and singly bound TSase with similar affinities, yet the two binding events have different enthalpy changes, providing further evidence of communication between the two active sites.
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Sapienza
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Lee
mesh_terms: D019943:Amino Acid Substitution; D003856:Deoxyuracil Nucleotides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003856:Deoxyuracil Nucleotides; D013940:Thymidylate Synthase