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Widespread Perturbation of Function, Structure, and Dynamics by a Conservative Single-Atom Substitution in Thymidylate Synthase
journal contribution
posted on 2016-09-20, 00:00 authored by Paul J. Sapienza, Andrew L. LeeThymidylate
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, for TSase at all
vertices of the dUMP binding/C146S mutation thermodynamic cycle and
found that the calculated TΔΔS°conf is 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 S2axis values
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.
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Escherichia coli TSasecovalent bondΔΔsingle-atom substitutionConservative Single-Atom SubstitutionS γWidespread Perturbationsulfur atomtitration calorimetryThymidylate Synthase Thymidylate synthaseCysteine 146reaction stepenzyme mechanismreaction mechanismresonance methyl symmetry axis order parametermutation causes perturbationsmeasure cofactor analog bindingS 2 axis valuescovalent bond formationentropic componentsheat signaturebinding eventsdrug targetdecreases substrate binding affinitychemical shiftsC 6enthalpy changesC 146S mutationC 146enthalpy changedUMP-bound states show