Mechanism of cis-prenyltransferase reaction probed by substrate analogues.
Undecaprenyl pyrophosphate synthase (UPPS) is a cis-type prenyltransferases which catalyzes condensation reactions of farnesyl diphosphate (FPP) with eight isopentenyl pyrophosphate (IPP) units to generate C(55) product. In this study, we used two analogues of FPP, 2-fluoro-FPP and [1,1-(2)H(2)]FPP, to probe the reaction mechanism of Escherichia coli UPPS. The reaction rate of 2-fluoro-FPP with IPP under single-turnover condition is similar to that of FPP, consistent with the mechanism without forming a farnesyl carbocation intermediate. Moreover, the deuterium secondary KIE of 0.985±0.022 measured for UPPS reaction using [1,1-(2)H(2)]FPP supports the associative transition state. Unlike the sequential mechanism used by trans-prenyltransferases, our data demonstrate E. coli UPPS utilizes the concerted mechanism.
Lu
Yen-Pin
YP
Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan, ROC.
Liu
Hon-Ge
HG
Teng
Kuo-Hsun
KH
Liang
Po-Huang
PH
D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D045782:Hemiterpenes / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D012717:Sesquiterpenes / Q000378:metabolism*; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*
C525013:2-fluorofarnesyl diphosphate; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004809:isopentenyl pyrophosphate; C004808:farnesyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C106821:cis-prenyl transferase; C023266:undecaprenyl pyrophosphate synthetase