THE MECHANISM OF PRENYLTRANSFERASE
tive cooperativity may be diagnostic of sequential conformational changes (Levitzki and Koshland, 1969). Acknowledgments The authors express their appreciation to Ruth M . Davis for her critical reading of the manuscript and to Evelyn Grace and Linda Laine for their competent typing. References Carminatti, H., JimCnez de Asila, L., Leiderman, B., and Rozengurt, E. (1971), J . Biol. Chem. 246, 7284. Ibsen, K. H., Murray, L., and Marles, S. W., Biochemistry (the preceding paper in this issue). Ibsen, K. H., and Trippet, P. (1972), Biochemistry 11, 4442. Ibsen, K. H., and Trippet, P. (1973), Arch. Biochem. Biophys. 156, 730. Ibsen, K. H., and Trippet, P. (1974), Arch. Biochem. Biophys. 163, 570. Ibsen, K. H., Trippet, P., and Basabe, J . R. (1975), Isozymes I. Molecular Structure, C. L. Markert, Ed., New York, N.Y., Academic Press, p 543. Irving, M. C., and Williams, J. F. (1973), Biochem. J . 131, 287.
JimCnez de A d a , L., Rozengurt, E., Devalle, J., and Carminatti, H. (1971), Biochim. Biophys. Acta 235, 326. Kayne, F. J., and Price, N. C . (1972), Biochemistry 11, 4415. Kayne, F. J., and Price, N. C. (1973), Arch. Biochem. Biophys. 159. 292. Levitzki, A., and Koshland, D. E., Jr. (1969), Proc. Natl. Acad. Sci. U.S.A. 62, 1121. Rozengurt, E., JimCnez de AsCa, L., and Carminatti, H . (1973), FEBS Lett. 31, 225. Schwark, W. S., Singhal, R. L., and Linz, C. M. (1971), J . Neurochem. 18, 123. Seubert, W., and Schoner, W . (1971); Curr. Top. Cell. Regul. 3, 237. Sparmann, G., Schulz, J., and Hofmann, E. (1973), FEBS Lett. 36, 305. Van Berkel, T. J. C. (1974), Biochim. Biophys. Acta 370, 140. Van Berkel, T. J. C., Koster, T. F., and Hulsmann, W. C . (1973), Biochim. Biophys. Acta 321, 171. Vijayvargiya, R., Schwark, W . S., and Singhal, R. L. (1969), Can. J . Biochem. 47, 895. Weber, G . (1969), Proc. Natl. Acad. Sci. U.S.A. 63, 1365. Wieker, H . J., Johannes, K. J., and Hess, B. (1973), Acta Biol. Med. Ger. 31, 259.
Prenyltransferase: The Mechanism of the Reaction? C . Dale Poulter*,j and Hans C. Rilling*
ABSTRACT: The enzyme, prenyltransferase, which normally catalyzes the addition of an allylic pyrophosphate to isopentenyl pyrophosphate, has been found to catalyze the hydrolysis of its allylic substrate. The rate of this hydrolysis is markedly stimulated by inorganic pyrophosphate. Competition experiments with 2-fluoroisopentenyl pyrophosphate and inorganic pyrophosphate demonstrated that inor-
ganic pyrophosphate stimulated hydrolysis by binding at the isopentenyl pyrophosphate site. Hydrolysis carried out in H2I80 or with (lS)-[l-3H]geranyl pyrophosphate show the C - 0 bond is broken and the C I carbon of geranyl pyrophosphate is inverted in the process. These results are interpreted to favor a carbonium ion mechanism for the prenyltransferase reaction.
Prenyltransferase (EC 2.5.1.1) catalyzes the condensation between Cd of isopentenyl and C I ’ of an allylic pyrophosphate, generating the five-carbon homologue of the allylic pyrophosphate. This condensation is the fundamental chain elongation reaction of terpene biosynthesis and leads to the
formation of such diverse products as sterols, carotenes, dolichols, and the hydrocarbon side chains of the respiratory coenzymes. The mechanisms which have been proposed for prenyl transfer can be grouped into two broad categories-those in which condensation is initiated by heterolytic cleavage of the carbon-oxygen bond of the allylic pyrophosphate, with or without assistance from the double bond of isopentenyl pyrophosphate, yielding cationic intermediates (Lynen et al., 1958; Rilling and Bloch, 1959; Cornforth and Popjak, 1959; Cornforth, 1968), and those in which condensation is initiated by attack of a nucleophilic group at the double bond of isopentenyl pyrophosphate with simultaneous formation of the Cl’-C4 bond between the two substrates and rupture of the CI’-oxygen bond (Cornforth et al., 1966: Cornforth, 1968). On the basis of the observation that C1‘ is inverted during prenyl transfer, Cornforth and Popjak ar-
4
A 4O
P
P Prenyitransferase
I R*Opp
T* R H+. PPI
From the Departments of Chemistry and Biochemistry, University of Utah, Salt Lake City, Utah 841 12. Received August 28, 1975. This investigation was supported by Research Grants G M 21328 and A M I3 I40 from the National Institutes of Health, f Alfred P. Sloan Fellow, 1975-1977; Career Development Award from the National Institutes of Health, H L 00084, 1975-1980.
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POULTER
gued that formation of a bond between C1' and C4 is concerted with cleavage of the CI'-oxygen bond (Cornforth et a]., 1966; Cornforth, 1968). They further postulated that the stereochemistry of proton elimination from C2 of isopentenyl pyrophosphate required the participation of a nucleophilic X group. The mechanism proposed by Cornforth and Popjak is commonly accepted as the mechanism for the prenyltransferase reaction. Recently, prenyltransferase from several sources has been purified to homogeneity (Eberhardt and Rilling, 1975: Reed and Rilling, 1975). During equilibrium dialysis experiments designed to measure the binding of substrate to the avian liver enzyme, Brent Reed in this laboratory observed that the enzyme also catalyzed the hydrolysis of the allylic substrate upon prolonged incubation. It seemed to us that examination of this novel catalytic activity of the enzyme might provide new information about the mechanism of prenyl transfer. In this paper we report the results of these experiments and present a mechanism for the reaction Materials and Methods
Porcine Licer Prenyltransferase. The enzyme was purified from pig liver by methods similar to those used for the preparation of avian liver enzyme and was assayed by the acid-lability method (Reed and Rilling, 1975). The protein was crystalline and homogeneous as judged by electrophoresis in natural and sodium dodecyl sulfate-containing polyacrylamide gels. The purification procedure will be published elsewhere. Substrates. [ 1-I4C]Isopentenyl pyrophosphate purchased from Amersham/Searle was diluted to a specific activity of I O pCi/wmol with synthetic material. [ 1 -3Hz]Geranyl pyrophosphate and geranyl pyrophosphate were prepared from the alcohols by the method of Cramer (Cramer and Weiman, 1960; Cornforth and Popjak, 1969). The pyrophosphate esters were purified by ion-exchange chromatography on Dowex 1 - X 8 formate. Columns ( 1 X 16 cm) were developed with 300 ml of a linear gradient of 0.1 1-0.63 M ammonium formate in methanol-water 90:lO. (1S)-[l-3H]Geraniol was prepared by the stereoselective reduction of [ 1 -3H]geraniol by yeast alcohol dehydrogenase and N A D H . The resulting alcohol was pyrophosphorylated and the product isolated as described above All other reagents used were the purest obtainable commercially. Hydrojysis of Geranyl Pyrophosphate. Prenyltransferase ( 1 3 pg, specific activity 580) and geranyl pyrophosphate (2 nmol, specific activity 120 pCi/wmol) were incubated at 37 " C in a mixture containing glycylglycine, pH 7.0, I O m M ; MgC12, 2 mM; mercaptoethanol, 1 m M ; and varying concentrations of inorganic pyrophosphate in a volume of 50 PI. A t five time intervals between 0 and 20 min, 1O-wl aliquots were removed and added to 1 ml of methanol containing 0.5% ammonia and 0.2-0.3 ml of Dowex I-X8 ion-exchange resin (formate form). After mixing, the samples were transferred to disposable Pasteur pipets which were plugged with cotton, and the eluates were collected in scintillation counting vials. The columns were washed with two 1-ml portions of methanol. A few drops of formic acid (68%) were added to the eluate followed by 20 ml of 0.4% Omnifluor (New England Nuclear) in toluene. Radioactivity was determined by liquid scintillation spectrometry. T h e linear portions of the velocity curve were used for calculating the rate of reaction.
1080
BIOCHEMISTRY, VOL.
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AND RlLLlNG
Table 1: Stimulation of Hydrolysis of Geranyl Pyrophosphate by Inorganic Pyrophosphate.
[Inorganic Pyrophosphate1
(M)
Rate of Geraniol Formationa (nmol min-' mg-9
0 4 x 10-6 1.2 x lo-' 2 x 10-5 4 x 10-5 2 x 6.7 x 2 x 10-3 6 x lo-' a
tive cooperativity may be diagnostic of sequential conformational changes (Levitzki and Koshland, 1969). Acknowledgments The authors express their appreciation to Ruth M . Davis for her critical reading of the manuscript and to Evelyn Grace and Linda Laine for their competent typing. References Carminatti, H., JimCnez de Asila, L., Leiderman, B., and Rozengurt, E. (1971), J . Biol. Chem. 246, 7284. Ibsen, K. H., Murray, L., and Marles, S. W., Biochemistry (the preceding paper in this issue). Ibsen, K. H., and Trippet, P. (1972), Biochemistry 11, 4442. Ibsen, K. H., and Trippet, P. (1973), Arch. Biochem. Biophys. 156, 730. Ibsen, K. H., and Trippet, P. (1974), Arch. Biochem. Biophys. 163, 570. Ibsen, K. H., Trippet, P., and Basabe, J . R. (1975), Isozymes I. Molecular Structure, C. L. Markert, Ed., New York, N.Y., Academic Press, p 543. Irving, M. C., and Williams, J. F. (1973), Biochem. J . 131, 287.
JimCnez de A d a , L., Rozengurt, E., Devalle, J., and Carminatti, H. (1971), Biochim. Biophys. Acta 235, 326. Kayne, F. J., and Price, N. C . (1972), Biochemistry 11, 4415. Kayne, F. J., and Price, N. C. (1973), Arch. Biochem. Biophys. 159. 292. Levitzki, A., and Koshland, D. E., Jr. (1969), Proc. Natl. Acad. Sci. U.S.A. 62, 1121. Rozengurt, E., JimCnez de AsCa, L., and Carminatti, H . (1973), FEBS Lett. 31, 225. Schwark, W. S., Singhal, R. L., and Linz, C. M. (1971), J . Neurochem. 18, 123. Seubert, W., and Schoner, W . (1971); Curr. Top. Cell. Regul. 3, 237. Sparmann, G., Schulz, J., and Hofmann, E. (1973), FEBS Lett. 36, 305. Van Berkel, T. J. C. (1974), Biochim. Biophys. Acta 370, 140. Van Berkel, T. J. C., Koster, T. F., and Hulsmann, W. C . (1973), Biochim. Biophys. Acta 321, 171. Vijayvargiya, R., Schwark, W . S., and Singhal, R. L. (1969), Can. J . Biochem. 47, 895. Weber, G . (1969), Proc. Natl. Acad. Sci. U.S.A. 63, 1365. Wieker, H . J., Johannes, K. J., and Hess, B. (1973), Acta Biol. Med. Ger. 31, 259.
Prenyltransferase: The Mechanism of the Reaction? C . Dale Poulter*,j and Hans C. Rilling*
ABSTRACT: The enzyme, prenyltransferase, which normally catalyzes the addition of an allylic pyrophosphate to isopentenyl pyrophosphate, has been found to catalyze the hydrolysis of its allylic substrate. The rate of this hydrolysis is markedly stimulated by inorganic pyrophosphate. Competition experiments with 2-fluoroisopentenyl pyrophosphate and inorganic pyrophosphate demonstrated that inor-
ganic pyrophosphate stimulated hydrolysis by binding at the isopentenyl pyrophosphate site. Hydrolysis carried out in H2I80 or with (lS)-[l-3H]geranyl pyrophosphate show the C - 0 bond is broken and the C I carbon of geranyl pyrophosphate is inverted in the process. These results are interpreted to favor a carbonium ion mechanism for the prenyltransferase reaction.
Prenyltransferase (EC 2.5.1.1) catalyzes the condensation between Cd of isopentenyl and C I ’ of an allylic pyrophosphate, generating the five-carbon homologue of the allylic pyrophosphate. This condensation is the fundamental chain elongation reaction of terpene biosynthesis and leads to the
formation of such diverse products as sterols, carotenes, dolichols, and the hydrocarbon side chains of the respiratory coenzymes. The mechanisms which have been proposed for prenyl transfer can be grouped into two broad categories-those in which condensation is initiated by heterolytic cleavage of the carbon-oxygen bond of the allylic pyrophosphate, with or without assistance from the double bond of isopentenyl pyrophosphate, yielding cationic intermediates (Lynen et al., 1958; Rilling and Bloch, 1959; Cornforth and Popjak, 1959; Cornforth, 1968), and those in which condensation is initiated by attack of a nucleophilic group at the double bond of isopentenyl pyrophosphate with simultaneous formation of the Cl’-C4 bond between the two substrates and rupture of the CI’-oxygen bond (Cornforth et al., 1966: Cornforth, 1968). On the basis of the observation that C1‘ is inverted during prenyl transfer, Cornforth and Popjak ar-
4
A 4O
P
P Prenyitransferase
I R*Opp
T* R H+. PPI
From the Departments of Chemistry and Biochemistry, University of Utah, Salt Lake City, Utah 841 12. Received August 28, 1975. This investigation was supported by Research Grants G M 21328 and A M I3 I40 from the National Institutes of Health, f Alfred P. Sloan Fellow, 1975-1977; Career Development Award from the National Institutes of Health, H L 00084, 1975-1980.
BIOCHEMISTRY, VOL.
15, NO. 5, 1976
1079
POULTER
gued that formation of a bond between C1' and C4 is concerted with cleavage of the CI'-oxygen bond (Cornforth et a]., 1966; Cornforth, 1968). They further postulated that the stereochemistry of proton elimination from C2 of isopentenyl pyrophosphate required the participation of a nucleophilic X group. The mechanism proposed by Cornforth and Popjak is commonly accepted as the mechanism for the prenyltransferase reaction. Recently, prenyltransferase from several sources has been purified to homogeneity (Eberhardt and Rilling, 1975: Reed and Rilling, 1975). During equilibrium dialysis experiments designed to measure the binding of substrate to the avian liver enzyme, Brent Reed in this laboratory observed that the enzyme also catalyzed the hydrolysis of the allylic substrate upon prolonged incubation. It seemed to us that examination of this novel catalytic activity of the enzyme might provide new information about the mechanism of prenyl transfer. In this paper we report the results of these experiments and present a mechanism for the reaction Materials and Methods
Porcine Licer Prenyltransferase. The enzyme was purified from pig liver by methods similar to those used for the preparation of avian liver enzyme and was assayed by the acid-lability method (Reed and Rilling, 1975). The protein was crystalline and homogeneous as judged by electrophoresis in natural and sodium dodecyl sulfate-containing polyacrylamide gels. The purification procedure will be published elsewhere. Substrates. [ 1-I4C]Isopentenyl pyrophosphate purchased from Amersham/Searle was diluted to a specific activity of I O pCi/wmol with synthetic material. [ 1 -3Hz]Geranyl pyrophosphate and geranyl pyrophosphate were prepared from the alcohols by the method of Cramer (Cramer and Weiman, 1960; Cornforth and Popjak, 1969). The pyrophosphate esters were purified by ion-exchange chromatography on Dowex 1 - X 8 formate. Columns ( 1 X 16 cm) were developed with 300 ml of a linear gradient of 0.1 1-0.63 M ammonium formate in methanol-water 90:lO. (1S)-[l-3H]Geraniol was prepared by the stereoselective reduction of [ 1 -3H]geraniol by yeast alcohol dehydrogenase and N A D H . The resulting alcohol was pyrophosphorylated and the product isolated as described above All other reagents used were the purest obtainable commercially. Hydrojysis of Geranyl Pyrophosphate. Prenyltransferase ( 1 3 pg, specific activity 580) and geranyl pyrophosphate (2 nmol, specific activity 120 pCi/wmol) were incubated at 37 " C in a mixture containing glycylglycine, pH 7.0, I O m M ; MgC12, 2 mM; mercaptoethanol, 1 m M ; and varying concentrations of inorganic pyrophosphate in a volume of 50 PI. A t five time intervals between 0 and 20 min, 1O-wl aliquots were removed and added to 1 ml of methanol containing 0.5% ammonia and 0.2-0.3 ml of Dowex I-X8 ion-exchange resin (formate form). After mixing, the samples were transferred to disposable Pasteur pipets which were plugged with cotton, and the eluates were collected in scintillation counting vials. The columns were washed with two 1-ml portions of methanol. A few drops of formic acid (68%) were added to the eluate followed by 20 ml of 0.4% Omnifluor (New England Nuclear) in toluene. Radioactivity was determined by liquid scintillation spectrometry. T h e linear portions of the velocity curve were used for calculating the rate of reaction.
1080
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NO.
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AND RlLLlNG
Table 1: Stimulation of Hydrolysis of Geranyl Pyrophosphate by Inorganic Pyrophosphate.
[Inorganic Pyrophosphate1
(M)
Rate of Geraniol Formationa (nmol min-' mg-9
0 4 x 10-6 1.2 x lo-' 2 x 10-5 4 x 10-5 2 x 6.7 x 2 x 10-3 6 x lo-' a
