Tryptophan 80 and Leucine 143 Are Critical for the Hydride Transfer Step of Thymidylate Synthase by Controlling Active Site Access
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- 作者：Timothy A. Fritz ; Lu Liu ; Janet S. Finer-Moore ; and Robert M. Stroud
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：June 4, 2002
- 年：2002
- 卷：41
- 期：22
- 页码：7021 - 7029
- 全文大小：678K
- 年卷期：v.41,no.22(June 4, 2002)
- ISSN：1520-4995
文摘
Mutant forms of thymidylate synthase (TS) with substitutions at the conserved active siteresidue, Trp 80, are deficient in the hydride transfer step of the TS reaction. These mutants produce a
mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-mercaptoethanol (mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-ME) adduct of the 2'-deoxyuridine-5'-monophosphate (dUMP) exocyclic methyleneintermediate. Trp 80 has been proposed to assist hydride transfer by stabilizing a 5,6,7,8-tetrahydrofolate(THF) radical cation intermediate [Barrett, J. E., Lucero, C. M., and Schultz, P. G. (1999) J. Am. Chem.Soc. 121, 7965-7966.] formed after THF changes its binding from the cofactor pocket to a putativealternate site. To understand the molecular basis of hydride transfer deficiency in a mutant in which Trp80 was changed to Gly, we determined the X-ray structures of this mutant Escherichia coli TS complexedwith dUMP and the folate analogue 10-propargyl-5,8-dideazafolate (CB3717) and of the wild-type enzymecomplexed with dUMP and THF. The mutant enzyme has a cavity in the active site continuous with bulksolvent. This cavity, sealed from bulk solvent in wild-type TS by Leu 143, would allow nucleophilicattack of mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-ME on the dUMP C5 exocyclic methylene. The structure of the wild-type enzyme/dUMP/THF complex shows that THF is bound in the cofactor binding pocket and is well positioned to transferhydride to the dUMP exocyclic methylene. Together, these results suggest that THF does not reorientduring hydride transfer and indicate that the role of Trp 80 may be to orient Leu 143 to shield the activesite from bulk solvent and to optimally position the cofactor for hydride transfer.