title: Rubber elongation by farnesyl pyrophosphate synthases involves a novel switch in enzyme stereospecificity.
abstract: A prenyltransferase purified from the commercial rubber tree, Hevea brasiliensis, that elongates existing cis-polyisoprene rubber molecules also catalyzes the formation of all trans-farnesyl pyrophosphate (t,t-FPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). In assays of the latter activity trans-geranyl pyrophosphate is the only other product identified. In contrast to this limited addition of IPP to DMAPP, we measured 7000 additions of isoprene per rubber molecule in a previous titration of active allylic ends of rubber molecules by purified prenyltransferase (Light, D. R., and Dennis, M. S. (1989) J. Biol. Chem. 264, 18589-18597). In order to confirm that purified prenyltransferase extensively elongates rubber molecules, doubly labeled [1-14C]isopentenyl [U-32P]pyrophosphate ([14C,32P]IPP) was synthesized. Using this reagent we show that both prenyltransferase purified from H. brasiliensis and prenyltransferase purified from avian liver (FPP synthase) add greater than 15 isoprene units to existing rubber molecules, consistent with the previous titration data. For confirmation that the prenyltransferase purified from H. brasiliensis adds isoprene units to rubber to make cis-polyisoprene, chirally tritiated [14C]IPP ([14C,2S-3H]IPP) was synthesized. Retention of the tritium label in FPP synthesized from [14C,2S-3H]IPP and DMAPP, geranyl pyrophosphate, or neryl pyrophosphate by prenyltransferase from H. brasiliensis or avian liver confirms trans addition to these substrates. In contrast, when [14C,2S-3H]IPP is incubated with serum-free rubber particles and prenyltransferase purified from H. brasiliensis, avian liver, or yeast, no tritium is incorporated into the rubber particles indicating cis addition. Thus, rubber particles have the ability to alter the stereoselective removal of the 2R-prochiral proton in favor of the removal of the 2S-prochiral proton. This apparent inversion of carbon 2 of IPP during the proton abstraction step by rubber particles represents a novel example of a switch in enzyme stereospecificity. In addition to being enzymatically similar to other prenyltransferases, rubber transferase also appears to be related immunologically to FPP synthases, since polyclonal antibodies to the H. brasiliensis prenyltransferase cross-react with the purified yeast prenyltransferase. In order to investigate potential primers of greater molecular weight than that of FPP, cis-undecaprenyl pyrophosphate (C55PP) was synthesized. C55PP stimulates the incorporation of [14C]IPP into rubber particles suggesting that it may prime new rubber molecules. However, in contrast to DMAPP, C55PP is not incorporated into any detectable products when incubated with prenyltransferase and [14C]IPP in the absence of rubber particles.(ABSTRACT TRUNCATED AT 400 WORDS)
authors: Department of Medicinal and Biomolecular Chemistry, Genentech, Inc., South San Francisco, California 94080.
authors: D R
authors: DR
authors: Light
authors: R A
authors: RA
authors: Lazarus
authors: M S
authors: MS
authors: Dennis
mesh_terms: D000818:Animals; D002645:Chickens; D004122:Dimethylallyltranstransferase / Q000378:metabolism*; D045782:Hemiterpenes; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D009943:Organophosphorus Compounds / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D010944:Plants / Q000201:enzymology*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D012408:Rubber / Q000378:metabolism*; D012717:Sesquiterpenes; D013237:Stereoisomerism; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*; D014197:Trees
chemical_list: D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D010710:Phosphates; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004809:isopentenyl pyrophosphate; C043060:3,3-dimethylallyl pyrophosphate; C015234:geranyl pyrophosphate; C004808:farnesyl pyrophosphate; D012408:Rubber; D014166:Transferases; D004122:Dimethylallyltranstransferase