In an effort to characterize the catalytic site of rubber transferase, the effects of two types of protein farnesyltransferase inhibitors, several chaetomellic acid A analogs 2, 4–7 and a-
Trang 1Protein farnesyltransferase inhibitors interfere with farnesyl
diphosphate binding by rubber transferase
Christopher J D Mau1, Sylvie Garneau2, Andrew A Scholte2, Jennifer E Van Fleet1, John C Vederas2 and Katrina Cornish1
1
USDA, Agricultural Research Service, Western Regional Research Center, Albany, CA, USA;2Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
Rubber transferase, a cis-prenyltransferase, catalyzes the
addition of thousands of isopentenyl diphosphate (IPP)
molecules to an allylic diphosphate initiator, such as farnesyl
diphosphate (FPP, 1), in the presence of a divalent metal
cofactor In an effort to characterize the catalytic site of
rubber transferase, the effects of two types of protein
farnesyltransferase inhibitors, several chaetomellic acid A
analogs (2, 4–7) and a-hydroxyfarnesylphosphonic acid (3),
on the ability of rubber transferase to add IPP to the allylic
diphosphate initiator were determined Both types of
com-pounds inhibited the activity of rubber transferases from
Hevea brasiliensis and Parthenium argentatum, but there
were species–specific differences in the inhibition of rubber transferases by these compounds Several shorter analogs
of chaetomellic acid A did not inhibit rubber transferase activity, even though the analogs contained chemical features that are present in an elongating rubber molecule These results indicate that the initiator-binding site in rubber transferase shares similar features to FPP binding sites in other enzymes
Keywords: Hevea brasiliensis; Parthenium argentatum; chaetomellic acid A; hydroxyfarnesylphosphonic acid
Rubber transferase catalyzes the biosynthesis of natural
rubber [1] To form this polymer of cis-polyisoprene, rubber
transferase adds up to thousands of molecules of
isopente-nyl diphosphate (IPP) to a single initiating allylic
diphos-phate, usually considered to be farnesyl diphosphate (FPP,
1, Fig 1) as the in vivo substrate However, rubber
transferase can also use other allylic diphosphates as
initiators; this substrate flexibility is probably a reflection
on the manner in which the catalytic site deals with the
elongating rubber polymer In addition, a divalent metal
cofactor, such as Mg2+, is required In spite of the
dependence of modern industrial society on natural rubber,
the biochemical properties of rubber transferase are only
partially understood [1–6]
Several compounds are known to bind to FPP sites in
other enzymes that use FPP as a substrate Most of these
substances have been discovered as a result of oncogenesis
studies involving protein farnesyltransferases
Chaetomel-lic acid A (2) (Fig 1), made by Chaetomella acutiseta, is an
inhibitor of protein farnesyltransferases (PFTs), such as
those that modify Ras, and competes for the FPP binding
site of PFTs with an IC50 of 55 nM [7] Derivatives of
chaetomellic acid A have also been found to inhibit PFTs [8] a-Hydroxyfarnesylphosphonic acid (HFPA, 3) (Fig 1)
is another compound shown to inhibit PFTs [9] with an
IC50of 30 nM[7]
In an effort to characterize the FPP binding site of rubber transferase, we have tested the ability of chaetomellic acid A and several analogs, as well as HFPA, to inhibit rubber biosynthesis in vitro We have used rubber transferases from Hevea brasiliensisand Parthenium argentatum to determine
if there are similarities in enzymatic behavior that might be characteristic of rubber transferases in general, as well as species-specific differences
Materials and methods Chemicals
Chemicals were purchased from Sigma Chemical Com-pany unless otherwise noted Farnesyl diphosphate (FPP), dimethyl allyl diphosphate (DMAPP) and [1-14C]IPP (2.04 GBqÆmmol)1) were purchased from American Radiolabe-led Chemicals, Inc (St Louis, MO, USA) a-Hydroxy-farnesylphosphonic acid (HFPA) was purchased from Calbiochem-Novabiochem Corp Washed rubber particles (WRP) from P argentatum and H brasiliensis were purified ([10,11], respectively) and stored in liquid nitrogen [12]
Synthesis of chaetomellic acid A analogs Several analogs of chaetomellic acid, purified as lithium salts, were made according to Ratemi et al [8] The structures of (Z)-2-octyl-3-methylbutenedioic acid dilithium
Correspondence to K Cornish, Western Regional Research Center,
USDA-ARS 800 Buchanan Street, Albany, CA 94710, USA.
Fax: + 1 510 559 5663, Tel.: + 1 510 559 5950,
E-mail: kcornish@pw.usda.gov
Abbreviations: DMAPP, dimethyl allyl diphosphate; FPP, farnesyl
diphosphate; IPP, isopentenyl diphosphate; HFPA,
a-hydroxyfarne-sylphosphonic acid; PFT, protein farnesyltransferase; WRP, washed
rubber particles; UDP, undecaprenyl diphosphate.
(Received 5 June 2003, revised 8 July 2003, accepted 31 July 2003)
Trang 2salt (4) (Z)-2-nerolyl-3-methylbutenedioic acid dilithium salt
(5) (Z)-2-farnesyl-3-methylbutenedioic acid dilithium salt
(6), and (Z)-2-geranyl-3-methylbutenedioic acid dilithium
salt (7) are shown in Fig 1 Concentrated stock solutions of
chaetomellic acid A analogs (4–7) were prepared in
dimethylsulfoxide at 2 mM
Reaction conditions
Rubber transferase assays were performed in multiwell
plates as described in Mau et al [13] The typical reaction
assay contained 1 mM [1-14C]IPP (7.03 MBqÆmmol)1),
1.25 mM MgSO4, 5 mM dithiothreitol and 100 mM Tris
pH 7.5 in a total volume of 50 lL Various concentrations
of initiators (FPP or DMAPP) were added, ranging from
10 pM to 1 mM, along with the stated concentrations of
chaetomellic acid A analogs (4–7) or HFPA (3) (that was
dissolved in ethanol) For rubber transferase assays (all
performed in triplicate), 0.5 mg of H brasiliensis WRP
was used; 0.25 mg of WRP was used per assay involving
P argentatum rubber transferase The reactions were
started by the addition of WRPs to the other components
and were incubated at 25°C for H brasiliensis WRP and
16°C for P argentatum WRP The assays were incubated
for 4 h and were stopped by the addition of 0.5MEDTA
pH 8 to a final concentration of 20 mM The incorporated
14C was measured by liquid scintillation counting of the
newly synthesized rubber which had been trapped on
filters and subsequently washed to remove unincorporated
[14C]IPP
Results Effects of various organic solvents and lithium salts
on rubber transferase activity Prior to the first assays involving the analogs, several organic solvents that could be used to dissolve the chaeto-mellic acid A analogs were added to the standard rubber transferase assay [13] to determine what effects the presence
of these solvents had on enzymatic activity The range of concentrations tested were typical working dilutions Dimethylsulfoxide and ethanol did not inhibit rubber transferase activity at the final concentration of 10% (v/v)
in the rubber transferase assay, so these were the chosen conditions for conducting the inhibitor studies (data not shown)
As the chaetomellic acid A analogs (4–7) were synthes-ized as lithium salts, the effect of lithium cations on rubber transferase activity was also evaluated The presence of LiCl
in the amounts of 1 lM to 1 mM did not affect rubber transferase activity (data not shown)
As a result, all subsequent assays involving analogs 4–7 were compared to internal controls containing comparable amounts of LiCl and dimethylsulfoxide Within any experiment in which the chaetomellic acid A analogs were diluted serially, the final dimethylsulfoxide concentration was kept constant at 10% Assays involving HFPA were compared to control reactions supplemented with ethanol, and serial dilutions of HFPA were made to maintain a final ethanol concentration of 10%
Fig 1 Structures of various chemicals tested for effects on rubber transferase activity in vitro Farnesyl diphosphate 1 is the presumed initi-ator in vivo Chaetomellic acid A (SG-2–29, 2) and a-hydroxyfarnesylphosphonic acid (HFPA, 3) are known inhibitors of protein farnesyltransferases, which covalently modify proteins with a FPP molecule (Z)-2-octyl-3-methylbutenedioic acid dilithium salt (SG-2–96, 4) (Z)-2-nerolyl-3-methyl-butenedioic acid dilithium salt (SG-1–27, 5) (Z)-2-farnesyl-3-methylbutenedioic acid dilithium salt (SG-1–29, 6), and (Z)-2-geranyl-3-methylbutenedioic acid dilithium salt (SG-1–30, 7) are analogs of chaetomellic acid A.
Trang 3Chaetomellic acid A analogs inhibit rubber transferase
Hevea brasiliensis Incubating H brasiliensis WRP with
20 lM chaetomellic acid A (2) and varying amounts of
DMAPP or FPP initiator in a rubber transferase enzymatic
assay demonstrated that chaetomellic acid A (2) was
inhi-bitory when present at 20-fold molar excess of DMAPP or
200-fold molar excess FPP (Fig 2) The related compound
4 inhibited H brasiliensis rubber transferase by 40% when
included at 180 lMin the presence of 10 nMFPP (data not
shown)
Compounds 5, 6 and 7 (Fig 1) were tested for inhibitory
effects on H brasiliensis rubber transferase in vitro
Com-pound 5 was made to resemble the first cis-elongation
product formed when using DMAPP as an initiator, while
compounds 6 and 7 were synthesized to resemble FPP and
geranyl diphosphate, respectively Compound 6 inhibited
activity by 25% when present in the assay at 180 lM in
1800-fold molar excess of FPP Compounds 5 and 7 were
not inhibitory when included in the assay at 180 lMin over
a million-fold molar excess of initiator
Parthenium argentatum All five chaetomellic acid A
ana-logs (2, 4, 5, 6 and 7) were tested using the rubber transferase
from guayule, P argentatum, a species phylogenetically
distant from H brasiliensis Compounds 2 and 6 reduced
rubber transferase activity by 27% (when added at 180 lM
in a 18,000-fold molar excess) and by 48% (when present
at 180 lM in a 180-fold molar excess), respectively
Compounds 4, 5 and 7 did not inhibit rubber transferase
activity under the conditions tested (supplemented in the
assay at 180 lMin over a million-fold molar excess)
Because compounds 2 and 6 were more inhibitory than 4
and 7, respectively, the long hydrophobic tails on these
molecules might have some important role in determining efficacy of inhibition To examine this possibility, palmitic acid and stearic acid were tested to see if either had any effect on rubber transferase activity When present at
180 lM, neither palmitate nor stearate inhibited IPP incor-poration into DMAPP- or FPP-initiated newly synthesized rubber by H brasiliensis or P argentatum (data not shown)
a-Hydroxyfarnesylphosphonic acid also inhibits rubber transferases As chaetomellic acid A had been described initially as an inhibitor of protein farnesyltransferase, another type of PFT inhibitor was also tested to determine
if it also could inhibit rubber transferase activity HFPA inhibited FPP utilization by H brasiliensis and P argent-atumWRP by 36–37% when present at 20 lMat 2000-fold molar excess
Determination of kinetic constants for chaetomellic acid
A analogs and HFPA Double reciprocal plots (1/v vs 1/[FPP]) of kinetic experiments indicated that compounds 2 and 3 were competitive inhibitors of H brasiliensis rubber transferase (Fig 3A,B), whereas compound 6 was a noncompetitive inhibitor (Fig 3C) In contrast, all three compounds behaved as competitive inhibitors of the
P argentatumrubber transferase (Fig 4A–C)
The apparent Kis for the chaetomellic acid A analogs and HFPA were determined from plots (not shown) of the slope
of the each reciprocal plot vs the concentration of the inhibitory compound [14] (Table 1)
Discussion Rubber transferases exhibit a considerable degree of toler-ance and can bind to a variety of different sizes of allylic diphosphate initiator molecules, at least up to solanesyl (C45) diphosphate (M H Chapman and K Cornish, unpublished data) Furthermore, the affinity of rubber transferase for the initiator increases with the size of the initiator, up to FPP (C15) in P argentatum and geranylger-anyl diphosphate (C20) in H brasiliensis As a result, a model was proposed for the rubber transferase active site, envisioning the presence of non–specific hydrophobic interactions, which increased the affinity for longer allylic diphosphate substrates [15] Nevertheless, it was uncertain if the chaetomellic acid analogs with the diacid groups could occupy the initiator-binding site with enough affinity to inhibit rubber biosynthesis
As seen in Fig 2, chaetomellic acid A (2) was able to interfere with IPP incorporation in the rubber transferase assay Almost 10-times more DMAPP than FPP was needed to overcome the same degree of inhibition by chaetomellic acid A (2) (Fig 2) The ability of DMAPP or FPP to displace chaetomellic acid A from the initiator-binding site in H brasiliensis rubber transferase paralleled the ninefold lower affinity of the enzyme for DMAPP vs FPP (Km,DMAPPof 13.2 and Km,FPPof 1.5 lM[1]) The length of the hydrophobic carbon tail also affected the inhibitory activity of the chaetomellic acid A analogs Compound 6, which has a farnesyl tail, was weakly inhibitory at 180 lM, in both species, whereas 7, which has a shorter geranyl tail, was not inhibitory at all
Fig 2 Chaetomellic acid A inhibits H brasiliensis rubber transferase.
Assays were performed using H brasiliensis WRP, 1 m M [14C]IPP, an
allylic diphosphate initiator, and either chaetomellic acid A (SG-2–29)
or its analog SG-2–96 DMAPP was varied in the presence of 20 l M
SG-2–29 (d) or 20 l M SG-2–96 (s), while various concentrations of
FPP were tested in combination with 20 l M SG-2–29 (.) The amount
of [ 14 C]IPP incorporated in these reactions was compared to control
reactions containing dimethylsulfoxide and LiCl instead of the
chaetomellic acid A analogs.
Trang 4Chaetomellic acid A (2) with a 14-carbon aliphatic chain strongly inhibited H brasiliensis rubber transferase at
20 lMwhile 4, with a similar structure but having a shorter 8-carbon tail, required 180 lMbefore it became inhibitory
On the other hand, P argentatum rubber transferase activity was only weakly inhibited by 180 lMchaetomellic acid A (2) and not at all by 180 lMcompound 4 In both cases, lengthening the hydrophobic surface of the analog added additional interactions, which increased the binding affinity of the inhibitor for the initiator-binding site The difference in the inhibition of H brasiliensis and P argent-atumrubber transferases by chaetomellic acid A indicated that there may be differences in the catalytic site geometry between the two species
However, a hydrophobic aliphatic tail simply attached to
a negative charge is not the sole cause of the observed inhibitory activity, because neither palmitic nor stearic acid was inhibitory in either species (data not shown) High affinity binding of substrates in the catalytic site appears to require two negatively charged oxygen atoms at one end of the molecule
These criteria are met by a-hydroxyfarnesylphosphonic acid (3), which did inhibit rubber transferase activity in both species That two types of protein farnesyltransferase inhibitors could also interfere with rubber transferase, a cis-prenyltransferase, indicates that the initiator binding site
of rubber transferase shares similarities with the FPP binding site of the protein farnesyltransferases
For P argentatum rubber transferase, compounds 2, 3 and 6 all appear to inhibit FPP binding (and subsequent IPP incorporation) competitively (Figs 4A–C) In contrast, although 2 and 3 both competitively inhibited the H bra-siliensis rubber transferase (Figs 3A,B), 6 acted in a noncompetitive manner (Fig 3C)
The calculated Kis show that the active sites of
H brasiliensisand P argentatum rubber transferases have
a higher affinity for 2 than for 6 (Table 1) While the allylic compound 6 more closely resembles the FPP initiator, compound 2 with its more flexible aliphatic backbone probably makes more extensive contact with the nonspecific hydrophobic surface lining the catalytic cavity [15] Alternatively, the difference in length of the inhibitors allows 2 to interact with the polyisoprene molecules within the rubber particle In both cases, the additional interactions result in tighter binding of 2 when compared to that of 6
The interpretation of the double reciprocal plots must be qualified because the rubber transferase active site is localized on the surface of a rubber particle [15] This proximity to the membrane monolayer covering the rubber particle would preclude access to the active site from certain directions during the in vitro reactions In addition, experi-mental manipulations of washed rubber particles can be challenging
The binding of competitive inhibitors and substrates are mutually exclusive, which is not the case for noncompetitive inhibitors Non-competitive inhibition results when binding
of an inhibitor at a second site prevents catalysis at the normal active site, without causing any changes in the binding kinetics at the active site The P argentatum rubber transferase can only bind 6 in a manner which affects the apparent binding at the initiator substrate site On the other
Fig 3 Chaetomellic acid A and SG-1–29 are inhibitors of H
brasil-iensis rubber transferase Double reciprocal plots of 1/v vs 1/[FPP]
were created using kinetic data from H brasiliensis rubber transferase
assays Rubber transferase assays were conducted in the presence of
H brasiliensis WRP, 1 m M [14C]IPP, and the indicated amounts of
FPP v is measured in units of lmol [14C]IPP incorporated per g dry
weight rubber per 4 h (A) Chaetomellic acid A (SG-2–29) was
inclu-ded at concentrations of 10 (.), 20 (s) or 50 (d) l M during the assay
while FPP was varied from 10 n M to 100 n M (B) HFPA was present at
10 (.), 20 (s), or 40 (d) l M while FPP was varied from 10 n M to
50 n M (c) SG-1–29 was added at final concentrations of 50 (.), 100
(s), or 200 (d) l M while FPP was varied from 10 n M to 100 n M
Trang 5hand, the noncompetitive inhibition by 6 of H brasiliensis rubber transferase may be caused by interference resulting from the additional binding of compound 6 at the IPP binding site, which is in the proximity of the allylic diphosphate binding site Competition between IPP and allylic diphosphate for the IPP binding sites has been observed at high substrate concentrations [1,16], and 6 was present at a range of 2500 to 10 000-fold molar excess to FPP in the assay (30- to 130-times the Kmfor FPP [17]) These results suggest that the spatial orientation between the IPP and initiator binding sites differs between the two rubber transferases, as well as the ability to bind 6 at other surfaces within the active site
The different behavior between the two rubber trans-ferases towards 6 should also be considered in light of the cooperative effects between IPP and FPP if one assumes that 6 is perceived as FPP by both enzymes In the range of concentrations of 6 incubated in the assays, the two rubber transferases exhibit different degrees of negative coopera-tivity, with the P argentatum enzyme showing the strongest effect [17] Under these conditions, binding of the first initiator molecule decreases the ability of a free FPP to displace the bound FPP (or elongating polymer) The ability
of the FPP-like compound 6 to bind noncompetitively at an additional location within the H brasiliensis active site may explain some of differences in the degree of negative cooperativity between the two enzymes Furthermore, the development of the P argentatum rubber transferase with a low Kmfor FPP (about 150-fold lower in concentration than the corresponding constant for H brasiliensis [17]) may also explain the difference in degree of negative cooperativity, because compound 6 can bind competitively into the initiator site of the P argentatum enzyme, unlike the noncompetitive binding in the vicinity of the weaker affinity initiator binding site of the H brasiliensis protein More experimentation to elucidate the mechanism of the negative cooperativity can be performed, but the basis for this behavior will probably only become apparent after the crystal structures of the rubber transferases have been determined
The differing behavior of the various compounds tested, both in the amount of compound needed to create an observable effect and the different types of inhibition effected, supports earlier kinetic data on cosubstrate effects among the species [16,17] Differences in binding constants, competitive effects, and in substrate activation exist between species [1,15–17] Thus, although the rubber transferases
Fig 4 SG-1–29 and HFPA are competitive inhibitors of P argentatum
rubber transferase Double reciprocal plots of 1/v vs 1/[FPP] were
created using kinetics data from P argentatum rubber transferase
assays Rubber transferase assays were conducted in the presence of
P argentatum WRP, 1 m M [ 14 C]IPP, and the indicated amounts of
FPP v is measured in vitro of lmol [ 14 C]IPP incorporated per g dry
weight rubber per 4 h (A) Chaetomellic acid A (SG-2–29) was added
at 50 (.), 100 (s), or 200 (d) l M while FPP was varied from 1.3 n M to
50 n M (B) Assays contained HFPA at concentrations of 100 (.), 200
(s), or 500 (d) l M while FPP was varied from 5 n M to 100 n M (C)
SG-1–29 was present at final concentrations of 50 (.), 100 (s), or 200
(d) l M while FPP was varied from 5 n M to 100 n M
Table 1 Kinetic constants determined for the interaction between cha-etomellic acid A analogs or a-hydroxyfarnesylphosphonic acid with
H brasiliensis and P argentatum rubber transferases.
Compound
H brasiliensis P argentatum
K i (l M )
Type of competitor K i (l M )
Type of competitor SG-2–29 (2) 42 competitive 8.8 competitive HFPA (3) 64 competitive 420 competitive SG-1–29 (6) 140 noncompetitive 25 competitive
Trang 6from different species share many commonalities, they are
not identical These differences may have resulted from
evolutionary divergence alone or in combination with the
development of a different cellular environment for the
rubber transferase in each species; H brasiliensis rubber
transferase is found on rubber particles in a free-flowing
latex in laticifers, while P argentatum rubber transferase
is located on intracellular rubber particles in the bark
parenchyma
The discovery that compounds known to bind to other
FPP binding sites can interact with the initiator binding site
of rubber transferases opens a new approach to modeling
the catalytic site of this enzyme in the absence of crystal
structures We have already used other biochemical and
physical studies to elucidate some features of the catalytic
site [1,18]
Recently, two crystal structures of undecaprenyl
diphosphate (UDP) synthase, a cis-prenyltransferase that
catalyzes the formation of a 55-carbon carrier for glycosyl
residues in peptidoglycan synthesis in bacteria, have been
published [19,20] Information from these structures may be
helpful in our efforts to model the rubber transferase active
site because both enzymes are cis-prenyltransferases with
proposed catalytic sites near the cytosolic surface of the
membrane The active site contains a cleft flanked by
hydrophobic amino acids that surrounds the aliphatic
backbone of the substrate [20] The chain length is
apparently regulated by the size of the active site, and
site-directed mutants, in which bulky, hydrophobic residues at
the distal end of the catalytic site have been converted to
alanine, can produce longer polyprenyl molecules Similar
mutagenesis of the avian FPP synthase catalytic site also
extends the size of the product formed [21] These structures
suggest that the rubber transferase has a hydrophobic
channel to direct the elongating biopolymer, in this case to
the rubber particle interior [1,15,18] Unlike the UDP
synthase and the FPP synthase, rubber transferase appears
to lack the bulky hydrophobic residues at the distal end of
the active site The normal mechanism for releasing the
rubber molecule from the enzyme has not been determined
Thus, the results presented here further support the
proposed model for the rubber transferase active site in
which the presence of non–specific hydrophobic interactions
increase the affinity for longer allylic diphosphate substrates
[1,15] These results also indicate that structural differences
do exist between the rubber transferases from evolutionarily
divergent species
Acknowledgements
We thank Dr R Krishnakumar at the Rubber Research Institute of
India for supplying the H brasiliensis latex as a source for WRP and
Dr Francis Nakayama at the US Water Conservation Laboratory in
Phoenix, AZ for maintaining and harvesting P argentatum plants for
isolation of the P argentatum WRP used in experiments described here.
We also acknowledge the help of Ms Mary H Chapman and Dr Javier
Castillo´n for isolating the H brasiliensis and P argentatum WRP used
in our experiments Ms Saima Kint and Dr Thomas McKeon kindly
provided palmitic and stearic acids for control experiments Part of this
work has been supported by the Natural Sciences and Engineering
Research Council of Canada and the Alberta Heritage Foundation for
Medical Research.
References
1 Cornish, K (2001) Similarities and differences in rubber bio-chemistry among plant species Phytobio-chemistry 57, 1123–1134.
2 Park, R.B & Bonner, J (1958) Enzymatic synthesis of rubber from mevalonic acid J Biol Chem 233, 340–343.
3 Berndt, J (1963) The Biosynthesis of Rubber pp 1–22 US Gov-ernment Research Report AD-601729.
4 Lynen, F (1969) Biochemical problems of rubber synthesis.
J Rubber Res Inst Malaya 21, 389–406.
5 Archer, B.L & Audley, B.G (1987) New aspects of rubber bio-synthesis Bot J Linnean Soc 94, 181–196.
6 Benedict, C.R., Madhavan, S., Greenblatt, G.A., Venkatachalam, K.V & Foster, M.A (1989) The enzymatic synthesis of rubber polymer in Parthenium argentatum Gray Plant Physiol 92, 816–821.
7 Gibbs, J.B., Pompliano, D.L., Mosser, S.D., Rands, E., Lingham, R.B., Singh, S.B., Scolnick, E.M., Kohl, N.E & Oliff, A (1993) Selective inhibition of farnesyl-protein transferase blocks ras processing in vivo J Biol Chem 268, 7617–7620.
8 Ratemi, E.S., Dolence, J.M., Poulter, C.D & Vederas, J.C (1996) Synthesis of protein farnesyltransferase and protein geranylger-anyltransferase inhibitors: Rapid access to chaetomellic acid A and its analogs J Org Chem 61, 6296–6301.
9 Pompliano, D.L., Rands, E., Schaber, M.D., Mosser, S.D., Anthony, N.J & Gibbs, J.B (1992) Steady-state kinetic mechan-ism of Ras farnesyl: protein transferase Biochemistry 31, 3800– 3807.
10 Cornish, K & Backhaus, R.A (1990) Rubber transferase activity in rubber particles of guayule Phytochemistry 29, 3809– 3813.
11 Siler, D.J & Cornish, K (1993) A protein from Ficus elastica rubber particles is related to proteins from Hevea brasiliensis and Parthenium argentatum Phytochemistry 32, 1097– 1102.
12 Cornish, K & Bartlett, D.L (1997) Stabilisation of particle integrity and particle bound cis-prenyltransferase activity in stored, purified rubber particles Phytochem Anal 8, 130–134.
13 Mau, C.J.D., Scott, D.J & Cornish, K (2000) Multiwell filtration system results in rapid, high-throughput rubber transferase microassay Phytochem Anal 11, 356–361.
14 Segel, I (1975) Enzyme Kinetics John Wiley and Sons, New York, NY.
15 Cornish, K (2001) Biochemistry of natural rubber, a vital raw material, emphasizing biosynthetic rate, molecular weight and compartmentalization, in evolutionarily divergent plant species Nat Prod Report 18, 182–189.
16 Castillo´n, J & Cornish, K (1999) Regulation of initiation and polymer molecular weight of cis-1,4-polyisoprene synthesized in vitro by particles isolated from Parthenium argentatum (Gray) Phytochemistry 51, 43–51.
17 Cornish, K., Castillo´n, J & Scott, D.J (2000) Rubber mole-cular weight regulation in vitro, in plant species that produce high and low molecular weights in vivo Biomacromolecules 1, 632–641.
18 Cornish, K., Wood, D.F & Windle, J.J (1999) Rubber par-ticles from four different species, examined by transmission electron microscopy and electron-paramagnetic-resonance spin labeling, are found to consist of a homogeneous rubber core enclosed by a contiguous, monolayer biomembrane Planta 210, 85–96.
19 Fujihashi, M., Zhang, Y.-W., Higuchi, Y., Li, X.-Y., Koyama, T.
& Miki, K (2001) Crystal structure of cis-prenyl chain elongating enzyme, undecaprenyl diphosphate synthase Proc Natl Acad Sci USA 98, 4337–4342.
Trang 720 Ko, T.-P., Chen, Y.-K., Robinson, H., Tsai, P.-C., Gao, Y.-G.,
Chen, A.P.-C., Wang, A.H.-J & Liang, P.-H (2001) Mechanism
of product chain length determination and the role of a flexible
loop in Escherichia coli undecaprenyl-pyrophosphate synthase
catalysis J Biol Chem 276, 47474–47482.
21 Tarshis, L.C., Proteau, P.J., Kellogg, B.A., Sacchettini, J.C & Poulter, C.D (1996) Regulation of product chain length by iso-prenyl diphosphate synthases Proc Natl Acad Sci USA 93, 15018–15023.