Light D R, Lazarus R A, Dennis M S
Department of Medicinal and Biomolecular Chemistry, Genentech, Inc., South San Francisco, California 94080.
J Biol Chem. 1989 Nov 5;264(31):18598-607.
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)
从商用橡胶树巴西橡胶树中纯化出的一种异戊二烯基转移酶，它能延长现有的顺式聚异戊二烯橡胶分子，还能催化由二甲基烯丙基焦磷酸（DMAPP）和异戊烯基焦磷酸（IPP）形成全反式法呢基焦磷酸（t,t-FPP）。在对后一种活性的测定中，反式香叶基焦磷酸是唯一鉴定出的其他产物。与向DMAPP中有限添加IPP形成对比的是，在之前通过纯化的异戊二烯基转移酶对橡胶分子的活性烯丙基末端进行的滴定中，我们测得每个橡胶分子有7000次异戊二烯的添加（莱特，D.R.，和丹尼斯，M.S.（1989年）《生物化学杂志》264卷，18589 - 18597页）。为了证实纯化的异戊二烯基转移酶能广泛延长橡胶分子，合成了双标记的[1 - 14C]异戊烯基[U - 32P]焦磷酸（[14C,32P]IPP）。使用这种试剂，我们表明从巴西橡胶树纯化的异戊二烯基转移酶和从禽肝纯化的异戊二烯基转移酶（FPP合酶）都能向现有的橡胶分子添加超过15个异戊二烯单元，这与之前的滴定数据一致。为了证实从巴西橡胶树纯化的异戊二烯基转移酶向橡胶添加异戊二烯单元以形成顺式聚异戊二烯，合成了手性氚标记的[14C]IPP（[14C,2S - 3H]IPP）。由巴西橡胶树或禽肝的异戊二烯基转移酶从[14C,2S - 3H]IPP和DMAPP、香叶基焦磷酸或橙花基焦磷酸合成的FPP中氚标记的保留，证实了对这些底物的反式加成。相反，当[14C,2S - 3H]IPP与无血清橡胶颗粒以及从巴西橡胶树、禽肝或酵母纯化的异戊二烯基转移酶一起孵育时，没有氚掺入橡胶颗粒，表明是顺式加成。因此，橡胶颗粒有能力改变对2R - 前手性质子的立体选择性去除，有利于去除2S - 前手性质子。在橡胶颗粒进行质子提取步骤期间IPP的碳2的这种明显反转代表了酶立体特异性转换的一个新例子。除了在酶学上与其他异戊二烯基转移酶相似外，橡胶转移酶在免疫学上似乎也与FPP合酶相关，因为针对巴西橡胶树异戊二烯基转移酶的多克隆抗体与纯化的酵母异戊二烯基转移酶发生交叉反应。为了研究分子量比FPP更大的潜在引物，合成了顺式十一异戊烯基焦磷酸（C55PP）。C55PP刺激[14C]IPP掺入橡胶颗粒，表明它可能引发新的橡胶分子。然而，与DMAPP不同的是，当在没有橡胶颗粒的情况下将C55PP与异戊二烯基转移酶和[14C]IPP一起孵育时，它不会掺入任何可检测的产物中。（摘要截短至400字）
