D221 in Thymidylate Synthase Controls Conformation Change, and Thereby Opening of the Imidazolidine

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• 作者：Carleton R. Sage ; Melissa D. Michelitsch ; Thomas J. Stout ; Donna Biermann ; Robert Nissen ; Janet Finer-Moore ; and Robert M. Stroud
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：September 29, 1998
• 年：1998
• 卷：37
• 期：39
• 页码：13893 - 13901
• 全文大小：260K
• 年卷期：v.37,no.39(September 29, 1998)
• ISSN：1520-4995

文摘

In thymidylate synthase (TS), the invariant residue Asp-221 provides the only side chain thathydrogen bonds to the pterin ring of the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. All mutantsof D221 except cysteine abolish activity. We have determined the crystal structures of two ternarycomplexes of the Escherichia coli mutant D221N. In a complex with dUMP and the antifolate 10-propargyl-5,8-dideazafolate (CB3717), dUMP is covalently bound to the active site cysteine, as usual.CB3717, which has no imidazolidine ring, is also bound in the usual productive orientation, but is lessordered than in wild-type complexes. The side chain of Asn-221 still hydrogen bonds to N3 of thequinazoline ring of CB3717, which must be in the enol form. In contrast, the structure of D221N with5-fluoro-dUMP and 5,10-methylene-5,6,7,8-tetrahydrofolate shows the cofactor bound in two partiallyoccupied, nonproductive binding sites. In both binding modes, the cofactor has a closed imidazolidinering and adopts the solution conformation of the unbound cofactor. In one of the binding sites, the pterinring is turned around such that Asn-221 hydrogen bonds to the unprotonated N1 instead of the protonatedN3 of the cofactor. This orientation blocks the conformational change required for forming covalentternary complexes. Taken together, the two crystal structures suggest that the hydrogen bond betweenthe side chain of Asp-221 and N3 of the cofactor is most critical during the early steps of cofactor binding,where it enforces the correct orientation of the pterin ring. Proper orientation of the cofactor appears tobe a prerequisite for opening the imidazolidine ring prior to formation of the covalent steady-stateintermediate in catalysis.