Different reaction mechanisms for cis- and trans-prenyltransferases |
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Authors: | Yen-Pin Lu Po-Huang Liang |
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Institution: | a Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan, ROC b Institute of Biological Chemistry, 128 Academia Rd. sec. 2, Academia Sinica, Taipei 115, Taiwan, ROC |
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Abstract: | Octaprenyl diphosphate synthase (OPPs) and undecaprenyl diphosphate synthases (UPPs) catalyze consecutive condensation reactions of farnesyl diphosphate (FPP) with 5 and 8 isopentenyl diphosphate (IPP) to generate C40 and C55 products with trans- and cis-double bonds, respectively. In this study, we used IPP analogue, 3-bromo-3-butenyl diphosphate (Br-IPP), in conjunction with radiolabeled FPP, to probe the reaction mechanisms of the two prenyltransferases. Using this alternative substrate with electron-withdrawing bromo group at the C3 position to slow down the condensation step, trapping of farnesol in the OPPs reaction from radiolabeled FPP under basic condition was observed, consistent with a sequential mechanism. In contrast, UPPs reaction yielded no farnesyl carbocation intermediate under the same condition with radiolabeled FPP and Br-IPP, indicating a concerted mechanism. Our data demonstrate the different reaction mechanisms for cis- and tran-prenyltransferases although they share the same substrates. |
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Keywords: | IPP isopentenyl diphosphate FPP farnesyl diphosphate GPP geranyl diphosphate FPPs farnesyl diphosphate synthase OPPs octaprenyl diphosphate synthase UPPs undecaprenyl diphosphate synthase UPP undecaprenyl diphosphate TLC thin layer chromatography FOH farnesol Br-IPP 3-Bromo-3-butenyl diphosphate Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid |
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