Partitioning Roles of Side Chains in Affinity, Orientation, and Catalysis with Structures for Mutant Complexes: Asparagine-229 in Thymidylate Synthase †,‡ Janet S. Finer-Moore,* ,§ Lu Liu, | Christian E. Schafmeister, ⊥ David L. Birdsall, § Ted Mau, ⊥ Daniel V. Santi, §,| and Robert M. Stroud § Department of Biochemistry and Biophysics, Department of Pharmaceutical Chemistry, and Graduate Group in Biophysics, UniVersity of California, San Francisco, San Francisco, California 94143-0448 ReceiVed NoVember 20, 1995; ReVised Manuscript ReceiVed February 16, 1996 X ABSTRACT: Thymidylate synthase (TS) methylates only dUMP, not dCMP. The crystal structure of TS‚dCMP shows dCMP 4-NH 2 excluded from the space between Asn-229 and His-199 by the hydrogen bonding and steric properties of Asn-229. Consequently, 6-C of dCMP is over 4 Å from the active site sulfhydryl. The Asn-229 side chain is prevented from flipping 180° to an orientation that could hydrogen bond to dCMP by a hydrogen bond network between conserved residues. Thus, the specific binding of dUMP by TS results from occlusion of competing substrates by steric and electronic effects of residues in the active site cavity. When Asn-229 is replaced by a cysteine, the Cys-229 Sγ rotates out of the active site, and the mutant enzyme binds both dCMP and dUMP tightly but does not methylate dCMP. Thus simply admitting dCMP into the dUMP binding site of TS is not sufficient for methylation of dCMP. Structures of nucleotide complexes of TS N229D provide a reasonable explanation for the preferential methylation of dCMP instead of dUMP by this mutant. In TS N229D‚dCMP, Asp-229 forms hydrogen bonds to 3-N and 4-NH 2 of dCMP. Neither the Asp-229 carboxyl moiety nor ordered water appears to hydrogen bond to 4-O of dUMP. Hydrogen bonds to 4-O (or 4-NH 2 ) have been proposed to stabilize reaction intermediates. If their absence in TS N229D‚dUMP persists in the ternary complex, it could explain the 10 4 -fold decrease in k cat /K m for dUMP. Mutation of key residues involved in binding reactants in transition states addresses the relative contributions of the residues both to enzymatic rate and to specific binding. Binding interactions of an enzyme with its biologically appropriate substrate rarely achieve the high affinity possible when the binding interactions are optimal. The binding of biotin by streptavidin is an example of binding that is closer to optimal, with ΔG f ∼ 21 kcal/M and K d ∼ 10 -15 M (Chaiet & Wolf, 1964). There are several reasons for this. Enzymes evolve to operate on the correct substrate at physiological concentrations. The difference between the observed binding energy and that theoretically possible can be turned toward enhancing catalytic rate, as seen in the increase in k cat with larger substrates in serine proteases, for example (Fersht, 1985). The enzyme binding site must also encode the lack of binding of other physiologically present molecules which would act as inhibitors or even as alternate substrates. This latter role is often overlooked in the search for a molecular basis for protein function. We probe this discriminatory role in mutants that alter preference in binding and catalysis. In the dUMP-methylating enzyme thymidylate synthase (TS), 1 Asn-229 makes two hydrogen bonds to the pyrimidine ring of dUMP and to that of the product, dTMP. It is the only side chain that is hydrogen bonded to the dUMP pyrimidine base in ternary complexes of TS, and the hydrogen bond it donates to 4-O interacts directly with the enolate formed in the reaction mechanism. Its key role in providing a donor to 4-O and an acceptor for 3-NH of dUMP and its conservation among all known TS sequences suggest it encodes specificity of the enzyme for dUMP versus other nucleotides. Indeed, TS does not methylate dCMP and binds this nucleotide with a K d ∼ 400-fold higher than that for dUMP (Liu & Santi, 1993a). Mutating Asn-229 to smaller residues that no longer provide hydrogen bonds to the pyrimidine ring has little effect on K d for dUMP, while several of these variants bind dCMP with a K d comparable to that of dUMP in wild-type TS (Liu & Santi, 1993a). dCMP is not a substrate for any of these mutants. However, mutation of Asn-229 to aspartate yields a TS variant that specifically methylates dCMP (Hardy & Nalivaika, 1992; Liu & Santi, 1992). To discover the structural basis for specific methylation of dUMP, and for the change in specificity that occurs when † This work was supported by NIH Grants CA-41323 to J.S.F.-M. and R.M.S. and CA-14394 to D.V.S. C.E.S. and T.M. are supported by Howard Hughes Medical Institute predoctoral fellowships. ‡ Coordinates for these structures are deposited in the Brookhaven Protein Data Bank. The access codes are 1NJA, 1NJB, 1NJC, 1NJD, and 1NJE. § Department of Biochemistry and Biophysics. | Department of Pharmaceutical Chemistry. ⊥ Graduate Group in Biophysics. X Abstract published in AdVance ACS Abstracts, April 1, 1996. 1 Abbreviations: TS, thymidylate synthase; dUMP, 2′-deoxyuridine 5′-monophosphate; dCMP, 2′-deoxycytidine 5′-monophosphate; dUrd, deoxyuridine; dCyd, deoxycytidine; CH2-H4folate, 5,10-methylene- 5,6,7,8-tetrahydrofolate; Fo, measured structure factor amplitude; Fc, calculated structure factor amplitude; (Fo - Fc)Rcalc map, electron density map calculated with coefficients (|Fo| - |Fc|) and phases calculated from the coordinates; (2Fo - Fc)Rcalc map, electron density map calculated with coefficients (2|Fo| - |Fc|) and phases calculated from the coordinates. 5125 Biochemistry 1996, 35, 5125-5136 0006-2960/96/0435-5125$12.00/0 © 1996 American Chemical Society