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The Purification of Prenyltransferase and Isopentenyl Pyrophosphate Isomerase of Pumpkin Fruit and Their Some Properties

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The Purification of Prenyltransferase and Isopentenyl Pyrophosphate Isomerase of Pumpkin Fruit and Their Some Properties The Journal of Biochemistry, Vol. 64, No. 2, 1968 The Purification of Prenyltransferase and Isopentenyl Pyrophosphate Isomerase of Pumpkin Fruit and Their Some Properties By KYOZO OGURA, TOKUZO NISHINO and SHUICHI SETO (From the Chemical Research Institute of Non-Aqueous Solutions, Tohoku University, Sendai) (Received for publication, March 2, 1968) Prenyltransferase (farnesyl pyrophosphate synthetase) [EC 2.5. 1. l] and isopentenylpyrophosphate isomerase [EC 5.3.3.2] were obtained and partially purified from pumpkin fruit. The prenyltransferase preparation catalyzed the condensation of isopentenyl pyrophosphate with dime thylallyl pyrophosphate as well as with geranyl pyrophosphate to yield trans-traps farnesyl pyrophosphate as a final product, and was free of isopentenyl pyrophosphate isomerase and geranylgeranyl pyrophosphate synthetase activities. Prenyltransferase of pumpkin has properties similar to those of pig liver, showing requirement of Mg++, Km value of 1.3•~ 10-6 M for geranyl pyrophosphate, and pH optimum at 7.5. Enzymatic formation of farnesyl pyro phatase [EC 3. 1.3. 11 of calf intestine was purchased from Boehringer and Soehne, Germany. Silica gel G phosphate which is an important precursor in the isoprenoid biosynthesis has been studied in and Kieselgur G were products of E. Merck, Germany. detail by using enzyme systems obtained from Geraniol, nerol and farnesol were generous gifts by Takasago Perfumary Co. Geranylgeraniol and geranyl yeast (1) and mammalian liver (2-6). It is linalool were kindly provided by Dr. Isler of Hoffmann known that farnesyl pyrophosphate synthetase - La Roche and Company, Switzerland. Sephadex G of pig liver catalyzes not only the reaction of 200 was a product of Pharmacia, Sweden. Hydro geranyl pyrophosphate with isopentenyl pyro xylapatite was prepared according to the method of phosphate but also the reaction of dimethylallyl TISELius et al. (11). Ion exchange resin was purchased pyrophosphate with isopentenyl pyrophosphate from Rhom and Hass, U.S.A. 1-14C-Isopentenyl pyro (3, 4). Isopentenyl pyrophosphate isomerase phosphate (1.2 ƒÊCi per ƒÊmole) was chemically synthe has also been isolated from the same organisms sized from methallyl chloride and 14C-barium carbonate (7-9). As to plant enzyme, however, only (Daiichi Pure Chemicals) essentially by the method of a single report of crude enzyme of carrot YUAN and BLOCH (12) except that the method of KANDUTSCH et al. (13) was employed for phosphoryl catalyzing the synthesis of geranylgeranyl ation of isopentenol. Purification of 14C-isopentenyl pyrophosphate is found in the literature, in pyrophosphate was performed by gradient elution on which only the reaction of farnesyl pyrophos a column of Amberlite CG-400 formate in a similar phate with isopentenyl pyrophosphate is des way described by BLOCH et al. (14). The product cribed (10). was identified by paper chromatography on Toyo filter The present paper reports the partial paper No. 51A in the system of n-propanol : ammonia : purification of prenyltransferase catalyzing water (6: 3: 1) (3, 10). Dimethylallyl, geranyl, and the formation of farnesyl pyrophosphate and farnesyl pyrophosphates were prepared by the pyro that of isopentenyl pyrophosphate isomerase phosphorylation of corresponding alcohols. Dimethyl from pumpkin fruit. Some of their enzymatic allyl alcohol was synthesized by the reduction of 3, 3- dimethylacrylic acid, which was synthesized from properties are described. mesity oxide, with lithium aluminium hydride (15). Assay for Isopentenyl Pyrophosphate Isomerase Activity MATERIALS AND METHODS -The reaction tubes contained, in a final volume of Pumpkins were obtained locally. Alkaline phos 1.0ml, 25ƒÊmoles of phosphate buffer, pH 7.0, 5ƒÊmoles 197 198 K. OGURA, T. NISHINO and S. SETO of magnesium chloride, 25 mpmoles of 14C-isopentenyl of 5-11% silicon oil DC-705 (Dow Corning Co.) on pyrophosphate (1.2 ƒÊCi per ƒÊmole) and 10-100 jug of base-washed 30-50 mesh firebrick was used for analysis enzyme protein. After the mixture had been incubated of C10-C15 alcohols. at 37•Ž for 40 min, the reaction was stopped by the For determination of optical densities a Cary addition of 0.2ml of 1 N hydrochloric acid , and the Recording spectrophotometer model 14 was used. mixture was kept at 37•Ž for 15 min to complete the hydrolysis of acid-labile dimethylallyl pyrophosphate RESULTS formed*. The mixture was made alkaline by addition Enzyme Purification-All steps were carried of a mixture of 0.3ml of 1 N sodium hydroxide and out at 4•Ž unless otherwise stated. A pumpkin 2.0ml of 2M sodium chloride . The mixture was extracted with 5ml of ethyl ether. After washing with (1 kg) was sliced, seeds being removed, pul water an aliquot (1.0ml)** of the extract was assayed verized in a homogenizer (Fuji juicer), and for radioactivity in toluene scintillator without evapo the homogenate was suspended in 500ml of ration of the solvent. For determination of radio 0.1 M phosphate buffer, pH 7.0. The mixture activity a Kobekogyo liquid scintillation counter model was squeezed through gauze, and the filtrate GSL-111 was used. was centrifuged at 40,000•~g for 1 hr. The Assay for Prenyllransferase (Farnesyl Pyrophosphate supernatant (670ml) was fractionated with Synthetase) Activity-The reaction tubes contained the ammonium sulfate, and the precipitated pro same mixture as that for the assay of isopentenyl tein between 40-65% saturation was dissolved pyrophosphate isomerase activity except that 25 mp moles of non-labeled dimethylallyl pyrophosphate or in a minimum volume of 0.05 M phosphate geranyl pyrophosphate was added. The activity was buffer. Both isomerase and prenyltransferase measured by the same method as that for the iso activities were contained in this 40-65% am pentenyl pyrophosphate isomerase activity except monium sulfate precipitated fraction. Table that ethyl ether was replaced by petroleum ether for I shows the enzymatic conversion of 14C-iso the extraction of radioactive materials. pentenyl pyrophosphate into acid-labile prenyl Thin Layer and Gas Chromatography-Thin layer pyrophosphates catalyzed by the crude fraction chromatography was performed in the two systems without any allylic pyrophosphate added. The described by McSWEENEY (16). In one system (nor resulting solution was placed on a column of mal phase) a plate coated with silica gel which had Sephadex G 200 which had been equilibrated been activated at 140•Ž for at least 1 hr , was deve with 0.01 M phosphate buffer, pH 6 .8, and loped with benzene: ethyl acetate (80:20) . In the eluted with the same buffer other method (reverse phase) a plate coated with . The effluent was Kieselgur G was activated at 110•Ž for 1 hr and then collected in 2ml portions, and each fraction impregnated with paraffin oil. After drying at 110•Ž for a few min, the plate was developed with acetone: TABLE I water (65:35) saturated with paraffin oil . Terpene alcohols for reference were located by exposing the Conversion of 14C-isopentenyl pyrophosphate developed plate to iodine vapor. After the plate was into acid-labile material. allowed to stand until the spots disappeared Each incubation mixture contained , sections , in a final of chromatographic plate were then scraped into volume of 1.0ml, 25 ƒÊoles of phosphate buffer , counting vials and the radioactive regions were deter pH 7.0 and 25 mƒÊmoles of 14C-isopentenyl pyro mined by liquid scintillation counting. Gas chro phosphate in addition to MgCl2 and enzyme as matography was carried out with a Hitachi gas indicated. Conditions of incubation and assay chromatograph type KGL-2A. A 2 m column of were the same as described in the section of assay butanediol succinate on Chromosorb W (Nikkaseiko Co .) for prenyltransferase activity . was used for analysis of C5 alcohols, and a 2m column * 14C-Isopentenyl pyrophosphate is so stabl e against this mild treatment with acid that it does not give any 14C-material extractable with ethyl ether or petroleum ether. ** The presence of 1 .0ml of ethyl ether or petro leum ether in toluene scintillator had very little quenching effect. Prenyltransferase and Isopentenyl-PP Isomerase 199 was assayed for both isopentenyl pyrophosphate The isomerase fractions (No. 12-17) and the isomerase and prenyltransferase activities. prenyltransferase fractions (No. 20-24) were Concentration of protein was followed by the combined respectively and they were used for measurement of optical densities at 280 mp. most studies. Prenyltransferase emerged a little earlier than Identificationof Products-Figure two shows isopentenyl pyrophosphate isomerase, but the the gas chromatographic separation of alcohols separation was not complete. The fractions liberated by acid treatment of the reaction containing the major part of both enzyme product formed by the incubation of 14C-iso activities were combined, and the combined pentenyl pyrophosphate with the isomerase fraction containing about 500 mg of protein fraction. The radioactivity trapped in the was chromatographed on a column of hydro effluent was present in carrier dimethylvinyl xylapatite (1.6•~16 cm) by elution with a linear carbinol and in carrier dimethylallyl alcohol, gradient of phosphate buffer, pH 6.8. The indicating the enzymatic formation of di mixing flask contained 150 ml of 0.01 M phos methylallyl pyrophosphate. It is well known phate buffer, pH 6.8, and the reservoir con that dimethylallyl pyrophosphate is readily tained 150 ml of 0.25 M phosphate buffer, pH hydrolyzed by the treatment with acid to yield 6.8. As seen in Fig. 1, prenyltransferase was a mixture of two isomeric alcohols, and that separated almost completely from isopentenyl the formation of the tertiary alcohol is pre pyrophosphate isomerase. The curve for the isomerase activity showed two peaks at fraction 9 and 14, but the analysis of the products by both fractions indicated that these two fractions catalyzed the isomerization of isopentenyl pyrophosphate to dimethylallyl pyrophosphate. Fio. 2. Gas chromatographic separation of alcohols liberated on acid treatment of the product of the enzymatic reaction.
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