(Received for publication, April 7, 1995; and in revised form, May 26, 1995 )
From the
Photoreactive analogues of prenyl diphosphates have been useful
in studying prenyltransferases. The effectiveness of analogues with
different chain lengths as probes of recombinant human protein
prenyltransferases is established here. A putative geranylgeranyl
diphosphate analogue, 2-diazo-3,3,3-trifluoropropionyloxy-farnesyl
diphosphate (DATFP-FPP), was the best inhibitor of both protein
farnesyltransferase (PFT) and protein geranylgeranyltransferase-I
(PGGT-I). Shorter photoreactive isoprenyl diphosphate analogues with
geranyl and dimethylallyl moieties and the DATFP-derivative of farnesyl
monophosphate were much poorer inhibitors. DATFP-FPP was a competitive
inhibitor of both PFT and PGGT-I with K values of 100 and 18 nM, respectively.
[
P]DATFP-FPP specifically photoradiolabeled the
-subunits of both PFT and PGGT-I. Photoradiolabeling of PGGT-I was
inhibited more effectively by geranylgeranyl diphosphate than farnesyl
diphosphate, whereas photoradiolabeling of PFT was inhibited better by
farnesyl diphosphate than geranylgeranyl diphosphate. These results
lead to the conclusions that DATFP-FPP is an effective probe of the
prenyl diphosphate binding domains of PFT and PGGT-I. Furthermore, the
-subunits of protein prenyltransferases must contribute
significantly to the recognition and binding of the isoprenoid
substrate.
Prenylated proteins have an important role in cellular
regulation, therefore, a description of their structure/function
relationships and post-translational processing has been of great
interest(1, 2, 3) . Among the
post-translational events is the modification of cysteine residues in
carboxyl termini with either a farnesyl or geranylgeranyl moiety by
sequence specific protein prenyltransferases. Protein
farnesyltransferase (PFT) ()is a
-heterodimer(4, 5) , which transfers the
C
isoprenoid from farnesyl diphosphate (FPP) to a Cys
residue in the sequence -Cys-A
-A
-Ser(Met, Gln),
where A
and A
represent aliphatic amino acids (1, 2, 3, 6, 7) . Protein
geranylgeranyltransferase-I (PGGT-I) is also a
-heterodimer(4) , but it transfers the C
moiety from geranylgeranyl diphosphate (GGPP) to a Cys residue in
proteins terminating in Leu
(-Cys-A
-A
-Leu)(8, 9, 10, 11) .
A different class of geranylgeranyltransferases, PGGT-II or rab
prenyltransferases(12) , modify proteins ending in -Cys-Cys,
-Cys-X-Cys, and
-Cys-Cys-X-X(8, 13, 14, 15) .
Although, the substrate specificity of prenyltransferases which
recognize the -C-A-A
-X motif has been
well established and many inhibitors of these enzymes have been
described(16, 17, 18, 19, 20) ,
the full extent of involvement of the protein subunits or their
respective amino acid residues in the prenylation reaction has not yet
been elucidated for any of these prenyltransferases. A detailed
description of the ``active sites'' of these enzymes would
aid in the development of more specific enzyme inhibitors based on
rational design. One means of investigating the structure of a
substrate binding domain is to probe it with photoreactive substrate
analogues. Earlier studies have shown photoinhibition and photolabeling
of bacterial prenyltransferases with a photoreactive analogue of FPP,
diazotrifluoropropionyloxy-geranyl diphosphate, DATFP-GPP (I, n=1),(21, 22) . DATFP-GPP was
subsequently shown to be an inhibitor of protein prenyltransferase
activity in cytosolic extracts of cultured human
lymphocytes(23) . DATFP-GPP and the GGPP analogue, DATFP-FPP (I, n=2), have now been used to probe protein
prenyltransferase function.
Since PFT and PGGT-I show specificity
toward both the protein and prenyl diphosphate substrates, the common
-subunit is not likely to be the sole determinant for substrate
specificity. Accordingly, Omer et al.(5) showed that
[
H]DATFP-GPP, the radiolabeled putative FPP
analogue, specifically labeled the
-subunit of recombinant human
PFT (hPFT). Recently Yokoyama et al.(24) have
synthesized [
H]DATFP-FPP and also shown that it
photoradiolabeled the
-subunit of bovine brain PGGT-I. Although,
the radioactive photoreactive prenyl diphosphate probes labeled only
the
-subunits of these prenyltransferases and the labeling could
be inhibited by the natural substrate for each enzyme, no evidence was
presented to unequivocally demonstrate that the photoprobe binds to the
active site. More specifically, demonstration that these probes inhibit
the prenyltransferases competitively, with K
values similar to the K
of the
natural substrates, was not established. In addition, neither
demonstration of chain length specificity of the photoprobes for
inhibition of activity was established nor was the specificity of the
prenyl diphosphates in their protection of the labeling of the
-subunits determined.
We present here an improved synthesis and
characterization of DATFP-FPP. DATFP-FPP was shown to competitively
inhibit both hPFT and hPGGT-I and demonstrated better inhibition than
the shorter DATFP-isoprenyl homologues. Furthermore, whereas
[P]DATFP-FPP (Fig.Z1) photolabeled the
-subunits of both human recombinant enzymes, inhibition of
radiolabeling showed preference for the enzyme's prenyl
diphosphate substrate.
Figure Z1: Structure 1
A critical step for this scheme is the selective
oxidation of (E,E)-3,7,11-trimethyl-1-(chloroacetoxy)-2,6,10-dodecatriene
(farnesyl chloroacetate) to (E,E)-3,7,11-trimethyl-1-(chloroacetoxy)-2,6,10-dodecatrien-12-ol
(-hydroxyfarnesyl chloroacetate, II). Farnesyl
chloroacetate, in contrast to geranyl chloroacetate (21) was
more reactive with selenious acid/t-butyl hydroperoxide. As a
result, the oxidation of trans
-terminal methyl group of
the farnesyl group was accompanied by the oxidation of methylene groups
as well. Increasing the extent of the reaction only led to the
formation of the over-oxidation products with more than one hydroxy or
carbonyl group in the molecule. Therefore, the conditions were
optimized for the oxidation by limiting consumption of the starting
material and minimizing the formation of over-oxidation products. This
was achieved by treatment for a shorter period of time and at a lower
temperature than previously reported(21, 27) .
Farnesyl chloroacetate (4.6 g, 15.5 mmol) was oxidized with t-butyl hydroperoxide (3.45 ml, 31 mmol) and
H
SeO
(1 g, 7.8 mmol) in 25 ml of
CH
Cl
for 2 h at 0 °C. The
-hydroxyfarnesyl chloroacetate was obtained in 24% yield (1.18 g,
3.8 mmol) after purification by silica gel chromatography with stepwise
gradient elution from petroleum ether/benzene (3:2, v/v) to
benzene/ethyl acetate (35:65, v/v). The alcohol eluted with 5-10%
ethyl acetate in benzene. Its R
was 0.32
in benzene/ethyl acetate (9:1, v/v). This yield represents a 6-fold
increase over the other reported synthesis(24) . Limited
consumption of the starting material also permitted its recovery for
subsequent utilization.
Standard one-pulse DQFCOSY and NOESY
experiments using a Varian Unity 600 nmr system were carried out to
establish that presumed structure (II) was correct. H nmr shifts
(ppm) (C
HCl
)
(600 MHz) were: 1.59 (3H, s), 1.65 (3H, s), 1.71 (3H, s), 1.98 (2H,
tr), 2.08 (2H, tr), 2.09 (4H, overlapping triplets), 3.96 (2H, s), 4.04
(2H, s), 4.69 (2H, d), 5.08 (1H, tr), 5.34 (1H, tr), 5.36 (1H, tr). The
DQFCOSY and NOESY analyses show that monohydroxylation of the terminal trans-methyl group was achieved. The presence of three methyl
peaks and four methylene signals in the spectrum rule out the
possibility that II was a secondary alcohol. The alcoholic group
obviously resides on the terminal carbon because the symmetrized
DQFCOSY spectrum showed that the methylene protons a (
,
3.9 ppm), assigned to the oxidized carbon, and the vinyl proton c (
, 5.36 ppm) were scalar coupled. The a protons were
coupled long range to the methylene protons d (
, 2.09
ppm) and the methyl group b (
, 1.65 ppm). The trans-orientation of the
-hydroxyl moiety was expected
by analogy to the work of others, who have established that
farnesylacetate (28) and other gem-dimethyl allylic
compounds (29) are oxidized by selenium dioxide with the
formation of the trans-hydroxy derivatives. This result was
confirmed here by noting that the chemical shifts of the vinyl proton c (
, 5.36) and the terminal methyl group b (
, 1.65) are the values expected for a trans-allylic
alcohol and are in contrast to the shifts (
, 5.25 (vinyl) and
, 1.78 (methyl)) expected for the corresponding cis alcohol (30) .
Other intermediates of the synthetic
pathway were pure as assessed by TLC using Kieselgel 60 F plates (E. Merck) and were analyzed by
H nmr analysis
using a Varian EM-390 (90 MHz) spectrometer. Their proton nmr spectral
results were consistent with expected products.
DATFP-farnesol was
phosphorylated by a previously described method (31) . The
products of phosphorylation, DATFP-FMP and DATFP-FPP, were separated by
DE52 cellulose (Whatman) column chromatography with a linear gradient
from 25 to 500 mM ammonium acetate in 50% (v/v) aqueous
methanol. DATFP-FPP was isolated in 10% yield (0.02 mmol) after further
purification by chromatography on CF-11 cellulose in tetrahydrofuran,
100 mM NHHCO
(85:15,
v/v)(25) . R
= 0.30 in
2-propanol/NH
OH/H
O (6:3:1, v/v/v).
= 11,600
L
mol
cm
(in 1 mM NH
OH).
Fractions containing DATFP-FMP were
lyophilized and applied to an Amberlite XAD-2 column in 1 mM aqueous ammonia. The pure monophosphate was eluted with 90%
methanol. R = 0.61 in
2-propanol/NH
OH/H
O (6:3:1, v/v/v).
= 9,700
L
mol
cm
(in 1 mM NH
OH).
Matrix-assisted Laser Desorption Ionization Mass
Spectrometry (MALDI-MS) of the isolated farnesylated peptide was
performed on a Voyager RP MALDI time-of-flight mass spectrometer
(PerSeptive Biosystems) in a positive ion analysis mode using
-cyano-4-hydroxycinnamic acid as a matrix.
Figure 1:
Inhibition of hPFT and hPGGT-I by
various DATFP-prenyl diphosphates. Assays were carried out as described
under ``Experimental Procedures'' using (A) hPFT, 2
ng/ml, with 50 nM [H]FPP and 10
µM Bt-KTKCVIS or (B) hPGGT-I, 1 ng/ml, with 45
nM [
H]GGPP, 1.5 µM Bt-KKFFCAIL, and different amounts of DATFP-FPP (▾),
DATFP-GPP (
), DATFP-DMAPP (
), and DATFP-FMP
(
).
Figure 2:
Double reciprocal plots of the activity of
hPFT and hPGGT-I in the presence of inhibitor DATFP-FPP. hPFT and
hPGGT-I were assayed as described under ``Experimental
Procedures'' using (A) 2 ng/ml recombinant hPFT, 10
µM Bt-KTKCVIS, variable [H]FPP, and
0 nM (
), 66.7 nM (
), and 200 nM (
) DATFP-FPP, and (B) 1.7 ng/ml of recombinant
hPGGT-I, 2 µM KKFFCAIL, variable
[
H]GGPP, and 0 nM (
), 22.2
nM (
), and 66.7 nM (
)
DATFP-FPP.
The
inhibition kinetics could also be a manifestation of the function of
the DATFP-derivatives as alternative substrates. Product formation from
the reaction of the DATFP-derivatives and peptide with hPFT was
assessed by two independent methods. No detectable product appearance
(or loss of KTKCVIS) was observed by HPLC analysis (see
``Experimental Procedures'' for details) when DATFP-FPP,
DATFP-GPP, or DATFP-DMAPP was used as an alternative prenyl donor. At
least 5 nmol of a prenylated peptide could have been detected by this
method of analysis. As a positive control, FPP and 25 nmol of
unbiotinylated KTKCVIS were incubated with hPFT over a 6-h period. A
time dependent loss of KTKCVIS (elution time = 17 min) and
appearance of a product peak (elution time = 27 min) were
observed. At the end of the incubation, the starting peptide was
completely consumed. The nature of the HPLC purified product was
established by MALDI-MS analysis. A parent molecular ion [M +
H] of 983 mass units, which corresponds to the expected mass for a
farnesylated peptide, was obtained. MALDI-MS analysis of a whole
incubation mixture without subsequent HPLC purification also showed the
appearance of farnesylated peptide. These experiments show that
DATFP-FPP cannot serve as a substrate in the reaction catalyzed by
hPFT, despite a low K value of hPFT for
DATFP-FPP. One concludes then that DATFP-FPP behaved as a dead end
inhibitor in the kinetic experiments shown on Fig.2A.
An alternative and more sensitive method of analysis was used to test
[
H]DATFP-GPP as a potential substrate for hPFT.
Incubation of hPFT with [
H]DATFP-GPP and
Bt-KTKCVIS showed no detectable radiolabeled product using the standard
enzymatic assay. The conclusions from this experiment support the
findings from HPLC analysis, but are tempered by the fact that
DATFP-GPP was not as good an inhibitor of hPFT as DATFP-FPP.
Figure 3:
Photolabeling of human PFT and PGGT-I with
[P]DATFP-FPP. hPFT (4 µg) or hPGGT-I (7
µg) were irradiated with 33.3 µM
[
P]DATFP-FPP (80 mCi/mmol) in 42 mM potassium phosphate buffer, pH 7.0, 3 mM MgCl
, 33 µM ZnCl
for 10 min
at 4 °C with or without Bt-peptide, FPP, or GGPP. Proteins were
separated by SDS-polyacrylamide gel electrophoresis in 10% Tris-Tricine
gels and silver stained (lanes 1-4). Labeled protein was
detected by autoradiography (12 h; lanes 1`-4`). Gels with
hPFT are in the leftpanels and those with hPGGT-I
are in the right panels. Irradiation conditions: lanes 1 and 1`, with 33 µM Bt-KTKCVIS and
Bt-KKFFCAIL, respectively; lanes 2 and 2`, without
the peptides; lanes 3 and 3`, with 67 µM FPP; lanes 4 and 4`, with 67 µM GGPP.
Figure 4:
Comparison of the inhibitory effect of
C- and C
-allylic diphosphates on the
photolabeling of the hPFT and hPGGT-I by
[
P]DATFP-FPP. hPFT (top) and hPGGT-I (bottom), 2 µg each, were irradiated for 15 min at 4
°C with 3.3 µM [
P]DATFP-FPP (1
Ci/mmol) in a 42 mM potassium phosphate buffer containing 3
mM MgCl
, 33 µM ZnCl
, and
different concentrations of FPP and GGPP. The concentration of FPP or
GGPP is shown below the graph. The
- (filled
bars) and
-subunits (open bars) were separated on a
10% Tris-Tricine polyacrylamide gel and counted for
radioactivity.
Weak but noticeable nonspecific
radiolabeling of the -subunits of the enzymes was also observed
(see Fig.3and Fig. 4), although the level of
nonspecific labeling varied from preparation to preparation of the
photoprobe. The level of
-subunit labeling was not affected by the
presence of isoprenoid substrates in photolysis incubations and it
appeared to be similar to that observed for the
-subunits under
conditions where the concentrations of inhibiting isoprenoid substrate
were very high.
The level of photolabeling of the -subunits of
both hPFT and hPGGT-I increased with increasing concentrations of
[
P]DATFP-FPP and was saturable (data not shown).
The apparent relative affinities of the prenyltransferases for
DATFP-FPP versus their natural prenyl diphosphate substrates
were also estimated indirectly by determining the dependence of
inhibition of the radiolabeling of the
-subunits on the
concentrations of FPP and GGPP. Prenyl diphosphate concentrations which
gave 50% inhibition of
-subunit photolabeling at 2.2
µM [
P]DATFP-FPP were 0.4 µM FPP and 1.1 µM GGPP, respectively, for hPFT and
hPGGT-I (data not shown). Therefore, the photoprobe binds well to both
enzymes but with an affinity which is poorer compared to FPP (for hPFT)
and comparable to GGPP (for hPGGT-I).
The application of photoaffinity labeling to the study of
prenyltransferases offers the possibility of exploring the active sites
of these enzymes. This points to the need to develop photoreactive
isoprenoid analogues which react specifically with different prenyl
diphosphate binding domains. Brems and Rilling (38) showed the
potential of using photoreactive isoprenoid analogues as probes of
prenyltransferases by demonstrating that
[H]o-azidophenylethyl diphosphate
labeled the isopentenyl diphosphate binding site of avian liver FPP
synthase. Our approach has been to synthesize analogues of the allylic
prenyl diphosphate substrate where the
-isoprene residue was
replaced by the photoreactive DATFP group. Previous work described the
synthesis of diazotrifluoropropionyloxy derivatives of dimethylallyl
and geranyl diphosphate (I, n = 0, 1) as
analogues of GPP and FPP, respectively(21) . The planar
configuration of the diazoacyl group was expected to mimic an isoprene
unit having a rigid arrangement of substituents around the double bond.
The effectiveness of DATFP-GPP as an inhibitory analogue of FPP has
been described for several
prenyltransferases(21, 22, 23, 39) .
Recently, [
H]DATFP-GPP and
[
H]DATFP-FPP have been used to photolabel the
-subunits of recombinant hPFT (5) and purified bovine
brain PGGT-I (24) , respectively.
We have now shown that both hPFT and hPGGT-I were strongly inhibited by DATFP-FPP, whereas the shorter chain analogues DATFP-GPP and DATFP-DMAPP were much poorer inhibitors. Furthermore, the monophosphate ester of the geranylgeranyl analogue, DATFP-FMP, was also a poor inhibitor. One would conclude then that the diphosphate moiety and a lipid moiety, at least as long as the DATFP-farnesyl group, are necessary for exhibiting strong inhibitory properties. Surprisingly, DATFP-FPP, the putative GGPP analogue, was a better inhibitor of hPFT than the FPP analogue DATFP-GPP. This might suggest that hPFT recognizes the farnesyl moiety of DATFP-FPP and binds it more tightly than the DATFP-geranyl moiety of DATFP-GPP.
The
apparent binding affinities of DATFP-FPP for hPFT and hPGGT-I were
estimated by two methods: 1) kinetic analysis of inhibition of
prenyltransferase activity by the photoprobe and 2) analysis of the
concentration-dependent inhibition of enzyme photolabeling by the
natural substrates FPP and GGPP. DATFP-FPP was a competitive inhibitor
of both hPFT and hPGGT-I. The K (100
nM) of hPFT for DATFP-FPP, determined kinetically, was 5 times
higher than the K
for FPP, whereas, the K
(18 nM) of hPGGT-I for
DATFP-FPP was about the same as the K
for
GGPP (Table1). The K
values
observed here are comparable to those previously reported for these
enzymes(5, 34) . Analyses of probe affinity for the
enzymes by the indirect method, where inhibition of photolabeling of
hPFT and hPGGT-I by FPP and GGPP was assessed, also showed that
relative differences in affinity between DATFP-FPP and the natural
substrates were similar to the ratios of K
/K
for
these compounds. Therefore, the affinities of DATFP-FPP for hPFT and
hPGGT-I were somewhat less than either of the natural substrates.
Nevertheless, DATFP-FPP binds competitively and tightly to both
enzymes.
The mode of involvement of protein subunits or their
respective amino acids residues in the active sites of
prenyltransferases in general has not been established, although the
first crystal structure for a prenyltransferase, FPP synthase, has now
been reported(40) . Radiolabeled photoanalogues of
prenyltransferase substrates have the potential to identify key
features of the protein structure of prenyltransferases. The FPP
analogue, [H]DATFP-GPP, has already been shown to
photolabel two other prenyltransferases(22, 41) in
addition to the
-subunits of hPFT (5) and
hPGGT-I(24) . The synthesis of the GGPP analogues,
[
H]- and [
P]DATFP-FPP, has
now made it possible to also photolabel purified bovine brain (24) and recombinant human PGGT-I. Heavy labeling of the
-subunits of these enzymes, in contrast to the weaker nonspecific
labeling of the
-subunits, points to the specificity of the
cross-linking procedure. Effective inhibition of labeling of the
respective prenyltransferases by their prenyl diphosphate substrates
and poor inhibition by the prenyl diphosphate substrate of the other
protein prenyltransferase clearly shows that the photoprobe is directed
toward the active site or ``substrate binding site'' and not
some other hydrophobic environment on the
-subunit. Therefore,
demonstrating that DATFP-FPP is a competitive inhibitor of the natural
allylic diphosphate substrates for the protein prenyltransferases and
showing that inhibition of labeling of the
-subunits of both hPFT
and hPGGT-I is specific for the prenyl diphosphate substrate leads to
the conclusion that the
-subunits of these protein
prenyltransferases are involved in the specific recognition and binding
of the prenyl diphosphate substrate. The mode of association of the
prenyl diphosphate with subunits of rab PGGT still remains to be
established.
The labeling of the -subunit with the isoprenoid
related probes is consistent with previous photo-cross-linking results
which showed that both peptide and protein substrates can interact with
the
-subunit of PFT(42, 43) . These probes now
offer the opportunity to identify specific amino acid residues in the
isoprenoid and peptide binding domains of these enzymes. The
P-labeled photoprobes offer some advantages over the
H-labeled probes for peptide localization and analysis
because they are more readily detectable. In addition, the
P-labeled probes can be easily prepared because
phosphorylation is the last step in the synthesis. Identification of
cross-linked amino acid residues would provide potential targets for a
variety of approaches to precisely define critical residues which are
involved in catalysis and determining the specificity of isoprenoid
substrate binding. Such approaches might include site-directed
mutagenesis and the design of other reactive prenyl diphosphate
analogues. Since both hPFT and hPGGT-I show a tolerance for alterations
in the structure of the
-isoprene unit when binding prenyl
diphosphate analogues, new isoprenoid based analogues might be designed
with more effective and site-specific functional groups attached at the
-terminus. One approach could be to incorporate substituents
which are chemically reactive with specific amino acid residues. One
can also envision the use of such photoprobes to map the binding sites
of other prenyltransferases and enzymes that metabolize prenyl
diphosphates. The synthesis of appropriate isoprenoid analogues could
bring new perspective to studies on proteins involved in prenylated
protein biosynthesis and function, such as COOH-terminal proteases,
methyl transferases, and prenylated protein binding proteins.