Probing the conformational change of Escherichia coli undecaprenyl pyrophosphate synthase during catalysis using an inhibitor and tryptophan mutants

Yi Hung Chen, Annie P.C. Chen, Chao Tsen Chen, Andrew H.J. Wang, Po Huang Liang

研究成果: 雜誌貢獻文章同行評審

39 引文 斯高帕斯(Scopus)

摘要

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation reactions of eight isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C 55 undecaprenyl pyrophosphate (UPP). In the present study, site-directed mutagenesis, fluorescence quenching, and stopped-flow methods were utilized to examine the substrate binding and the protein conformational change. (S)-Farnesyl thiopyrophosphate (FsPP), a FPP analogue, was synthesized to probe the enzyme inhibition and events associated with the protein fluorescence change. This compound with a much less labile thiopyrophosphate shows K i value of 0.2 μM in the inhibition of Escherichia coli UPPS and serves as a poor substrate, with the k cat value (3.1 × 10 -7 S -1) 10 7 times smaller than using FPP as the substrate. Reduction of protein intrinsic fluorescence was observed upon addition of FPP (or FsPP) to the UPPS solution. Moreover, fluorescence studies carried out using W91F and other mutant UPPS with Trp replaced by Phe indicate that FPP binding mainly quenches the fluorescence of Trp-91, a residue in the α3 helix that moves toward the active site during substrate binding. Using stopped-flow apparatus, a three-phase protein fluorescence change with time was observed by mixing the E·FPP complex with IPP in the presence of Mg 2+. However, during the binding of E·FsPP with IPP, only the fastest phase was observed. These results suggest that the first phase is due to the IPP binding to E·FPP complex, and the other two slow phases are originated from the protein conformational change. The two slow phases coincide with the time course of FPP chain elongation from C 15 to C 55 and product release.

原文英語
頁(從 - 到)7369-7376
頁數8
期刊Journal of Biological Chemistry
277
發行號9
DOIs
出版狀態已發佈 - 3月 1 2002
對外發佈

ASJC Scopus subject areas

  • 生物化學
  • 分子生物學
  • 細胞生物學

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