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)
Highlights
A prenyltransferase purified from the commercial rubber tree, Hevea bradiensis, that elongates existing cis-polyisoprene rubber molecules catalyzes the formation of all tram-farnesyl pyrophosphate
Using this reagent we show that both prenyltransferase purified from H. bra&lien&s and prenyltransferase purified from avian liver (FPP synthase) add >15 isoprene units to existing rubber molecules, consistent with the previous titration data
isopentenyl pyrophosphate (IPP) incorporation into rubber when added to latex or to drolyzed there were >1600 additional isoprene units added serum-free rubber particles and purified Heuea prenyltrans- per low molecular weight allylic pyrophosphate hydrolyzed in ferase or avian FPP synthase
Summary
A 90-g cell paste of E. coli W3110,washed and resuspended in 0.2 liter of 30 mM Tris, pH 8.0, was treated with 50 mg of eggwhite lysozyme (Sigma) and 31 mg of DTT for 20 min a t 30 "C and was cooled on ice after the addition of 10 mg of deoxyribonucleaseI (Sigma).The cell suspension converted to [2-3H]IPP and used to determine the stereo- was distributed by sonic oscillation on ice twice for 3 min at the chemistry of prenyltransferase proton removal in a study of farnesyl pyrophosphate (FPP)synthesis in low speed supernatant fluids of rat liver and yeast cell homogenates. After addition of 1.5ml of 2 M NaCl, the hydrolyzedproducts were extracted with 5 mlof hexane, and 4 mlof the organic phase was dried in scintillation vials for 2 h in a vacuum oven (80 "C) to remove low molecular weight radioactive compounds before addition of the scin-
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