4-Oxalocrotonate Tautomerase, Its Homologue YwhB, and Active Vinylpyruvate Hydratase: Synthesis and Evaluation of 2-Fluoro Substrate Analogues
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- 作者：William H. Johnson ; Jr. ; Susan C. Wang ; Thanuja M. Stanley ; Robert M. Czerwinski ; Jeffrey J. Almrud ; Gerrit J. Poelarends ; Alexey G. Murzin ; and Christian P. Whitman
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：August 17, 2004
- 年：2004
- 卷：43
- 期：32
- 页码：10490 - 10501
- 全文大小：343K
- 年卷期：v.43,no.32(August 17, 2004)
- ISSN：1520-4995
文摘
A series of 2-fluoro-4-alkene and 2-fluoro-4-alkyne substrate analogues were synthesized andexamined as potential inhibitors of three enzymes: 4-oxalocrotonate tautomerase (4-OT) and vinylpyruvatehydratase (VPH) from the catechol meta-fission pathway and a closely related 4-OT homologue found inBacillus subtilis designated YwhB. All of the compounds were potent competitive inhibitors of 4-OTwith the monocarboxylated 2E-fluoro-2,4-pentadienoate and the dicarboxylated 2E-fluoro-2-en-4-ynoatebeing the most potent. Despite the close mechanistic and structural similarities between 4-OT and YwhB,these compounds were significantly less potent inhibitors of YwhB with Ki values ranging from 5- to633-fold lower than those determined for 4-OT. The study of VPH is complicated by the fact that theenzyme is only active as a complex with the metal-dependent 4-oxalocrotonate decarboxylase (4-OD),the enzyme following 4-OT in the catechol meta-fission pathway. A structure-based sequence analysisidentified 4-OD as a member of the fumarylacetoacetate hydrolase (FAH) superfamily and implicatedGlu-109 and Glu-111 as potential metal-binding ligands. Changing these residues to a glutamine verifiedtheir importance for enzymatic activity and enabled the production of soluble E109Q4-OD/VPH or E111Q4-OD/VPH complexes, which retained full hydratase activity but had little decarboxylase activity. Subsequentincubation of the E109Q4-OD/VPH complex with the substrate analogues identified the 2E and 2Z isomersof the monocarboxylated 2-fluoropent-2-en-4-ynoate as competitive inhibitors. The combined results setthe stage for crystallographic studies of 4-OT, YwhB, and VPH using these inhibitors as ligands.