Widespread perturbation of function, structure, and dynamics by a conservative single-Atom substitution in thymidylate synthase

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°bind is 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,...