1bo8
THYMIDYLATE SYNTHASE R178T MUTANT
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OverviewOverview
In thymidylate synthase, four conserved arginines provide two hydrogen, bonds each to the oxygens of the phosphate group of the substrate, 2'-deoxyuridine-5'-monophosphate. Of these, R23, R178, and R179 are far, removed from the site of methyl transfer and contribute to catalysis, solely through binding and orientation of ligands. These arginines can be, substituted by other residues, while still retaining more than 1% activity, of the wild-type enzyme. We compared the kinetics and determined the, crystal structures of dUMP complexes of three of the most active, uncharged single mutants of these arginines, R23I, R178T, and R179T, and, of double mutants (R23I, R179T) and (R178T, R179T). The dramatically, higher K(m) for R178T compared to the other two single mutants arises from, the effects of R178 substitution on the orientation of dUMP; 10-15-fold, increases in for R23I and R178T reflect the role of these residues in, stabilizing the closed conformation of TS in ternary complexes. The free, energy for productive dUMP binding, DeltaG(S), increases by at least 1, kcal/mol for each mutant, even when dUMP orientation and mobility in the, crystal structure is the same as in wild-type enzyme. Thus, the four, arginines do not contribute excess positive charge to the PO(4)(-2), binding site; rather, they ideally complement the charge and geometry of, the phosphate moiety. More-than-additive increases in DeltaG(S) seen in, the double mutants are consistent with quadratic increases in DeltaG(S), predicted for deviations from ideal electrostatic interactions and may, also reflect cooperative binding of the arginines to the phosphate, oxygens.
About this StructureAbout this Structure
1BO8 is a Single protein structure of sequence from Lactobacillus casei with K and U as ligands. Active as Thymidylate synthase, with EC number 2.1.1.45 Full crystallographic information is available from OCA.
ReferenceReference
Energetic contributions of four arginines to phosphate-binding in thymidylate synthase are more than additive and depend on optimization of "effective charge balance"., Morse RJ, Kawase S, Santi DV, Finer-Moore J, Stroud RM, Biochemistry. 2000 Feb 8;39(5):1011-20. PMID:10653645
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