Partitioning Roles of Side Chains in Affinity, Orientation, and Catalysis with Structures for Mutant Complexes: Asparagine-229 in Thymidylate Synthase
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- 作者：Janet S. Finer-Moore ; Lu Liu ; Christian E. Schafmeister ; David L. Birdsall ; Ted Mau ; Daniel V. Santi ; and Robert M. Stroud
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：April 23, 1996
- 年：1996
- 卷：35
- 期：16
- 页码：5125 - 5136
- 全文大小：764K
- 年卷期：v.35,no.16(April 23, 1996)
- ISSN：1520-4995
文摘
Thymidylate synthase (TS) methylates only dUMP, not dCMP. Thecrystal structure ofTS·dCMP shows dCMP 4-NH2 excluded from the space betweenAsn-229 and His-199 by the hydrogenbonding and steric properties of Asn-229. Consequently, 6-C ofdCMP is over 4 Å from the active sitesulfhydryl. The Asn-229 side chain is prevented from flipping180
to an orientation that could hydrogenbond to dCMP by a hydrogen bond network between conserved residues.Thus, the specific binding ofdUMP by TS results from occlusion of competing substrates by steric andelectronic effects of residuesin the active site cavity. When Asn-229 is replaced by a cysteine,the Cys-229 S
rotates out of theactive site, and the mutant enzyme binds both dCMP and dUMP tightly butdoes not methylate dCMP.Thus simply admitting dCMP into the dUMP binding site of TS is notsufficient for methylation of dCMP.Structures of nucleotide complexes of TS N229D provide areasonable explanation for the preferentialmethylation of dCMP instead of dUMP by this mutant. In TSN229D·dCMP, Asp-229 forms hydrogenbonds to 3-N and 4-NH2 of dCMP. Neither the Asp-229carboxyl moiety nor ordered water appears tohydrogen bond to 4-O of dUMP. Hydrogen bonds to 4-O (or4-NH2) have been proposed to stabilizereaction intermediates. If their absence in TS N229D·dUMPpersists in the ternary complex, it couldexplain the 104-fold decrease inkcat/Km fordUMP.
to an orientation that could hydrogenbond to dCMP by a hydrogen bond network between conserved residues.Thus, the specific binding ofdUMP by TS results from occlusion of competing substrates by steric andelectronic effects of residuesin the active site cavity. When Asn-229 is replaced by a cysteine,the Cys-229 S rotates out of theactive site, and the mutant enzyme binds both dCMP and dUMP tightly butdoes not methylate dCMP.Thus simply admitting dCMP into the dUMP binding site of TS is notsufficient for methylation of dCMP.Structures of nucleotide complexes of TS N229D provide areasonable explanation for the preferentialmethylation of dCMP instead of dUMP by this mutant. In TSN229D·dCMP, Asp-229 forms hydrogenbonds to 3-N and 4-NH2 of dCMP. Neither the Asp-229carboxyl moiety nor ordered water appears tohydrogen bond to 4-O of dUMP. Hydrogen bonds to 4-O (or4-NH2) have been proposed to stabilizereaction intermediates. If their absence in TS N229D·dUMPpersists in the ternary complex, it couldexplain the 104-fold decrease inkcat/Km fordUMP.