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J. Biol. Chem., Vol. 277, Issue 9, 7369-7376, March 1, 2002

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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, and Po-Huang Liang^**

From the  Institute of Biological Chemistry, Academia Sinica, Taipei 11529 and  Institute of Biochemical Sciences and ^§ Department of Chemistry, National Taiwan University, Taipei 10098, Taiwan

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation reactions of eight isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C₅₅ 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 × 10⁷ s¹) 10⁷ 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 3 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 Mg²⁺. 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 C₁₅ to C₅₅ and product release.

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