(Received for publication, October 29, 1996, and in revised form, November 8, 1996)
From the Departments of Biochemistry and
§ Molecular Cancer Biology, Duke University Medical Center,
Durham, North Carolina 27710
Protein farnesyltransferase (FTase) is a zinc
metalloenzyme that catalyzes the addition of a farnesyl isoprenoid to a
conserved cysteine in peptide or protein substrates. We have
substituted the essential Zn2+ in FTase with
Co2+ to investigate the function of the metal polyhedron
using optical absorption spectroscopy. The catalytic activity of FTase
is unchanged by the substitution of cobalt for zinc. The absorption
spectrum of Co2+-FTase displays a thiolate-Co2+
charge transfer band (320 = 1030 M
1 cm
1) consistent with the
coordination of one cysteine side chain and also ligand field bands
(
560 = 140 M
1
cm
1) indicative of a pentacoordinate or distorted
tetrahedral metal geometry. Most importantly, the ligand-metal charge
transfer band displays an increased intensity (
320 = 1830 M
1 cm
1) in the ternary
complex of FTase·isoprenoid·peptide substrate indicative of the
formation of a second Co2+-thiolate bond as cobalt
coordinates the thiolate of the peptide substrate. A similar increase
in the ligand-metal charge transfer band in a product complex indicates
that the sulfur atom of the farnesylated peptide also coordinates the
metal. Transient kinetics demonstrate that thiolate-cobalt metal
coordination also occurs in an active FTase·FPP·peptide substrate
complex and that the rate constant for the chemical step is 17 s
1. These data provide evidence that the zinc ion plays
an important catalytic role in FTase, most likely by activation of the
cysteine thiol of the protein substrate for nucleophilic attack on the isoprenoid.
Protein farnesyltransferase (FTase)1 catalyzes the transfer of the farnesyl group of farnesyl pyrophosphate (FPP) to a conserved cysteine residue of a protein substrate, including Ras proteins, nuclear lamins, and several proteins involved in cell signaling (1, 2, 3, 4). Protein farnesylation mediates membrane association and, possibly, interactions with other proteins essential for the localization and function of these proteins (3, 4). One example in this regard is the requirement of farnesylation for the cell transforming ability of oncogenic Ras proteins (5); this result has stimulated an intense search for FTase inhibitors as potential anticancer drugs (6, 7). An increased understanding of the molecular mechanism of FTase should enhance the rational design of such FTase inhibitors.
FTase is a metalloenzyme that contains one zinc ion per /
heterodimer that is essential for optimal activity (8, 9). Cross-linking and direct binding studies indicate that the zinc ion is
required for the binding of protein but not isoprenoid substrates (8).
Additionally, FTase containing Cd2+ substituted for
Zn2+ has essentially normal catalytic activity,
demonstrating that other metal ions can functionally substitute for the
zinc (10). Chemical modification and site-directed mutagenesis studies
have identified a conserved cysteine residue of FTase,
Cys299, in the
subunit, as important for catalytic
activity and zinc binding, suggesting that the thiolate of this residue
may directly coordinate the zinc ion (11).
Although it is clear that the zinc ion in FTase is critical for activity, the precise function of the metal, particularly the question of whether the primary role of the zinc ion is structural or catalytic, is not yet known. Proposed catalytic functions for the zinc ion in FTase include increasing the nucleophilicity of the cysteine residue of the protein substrate (3, 8, 11, 12) and/or activating the diphosphate leaving group (11, 13, 14). Here we investigate the metal coordination polyhedron in FTase by substituting Co2+ for Zn2+, which does not change the catalytic activity of the enzyme. This substitution provides a useful spectroscopic probe of the composition and geometric arrangement of the ligands around the metal ion (15, 16), and the spectral data obtained indicate that the metal ion coordinates the thiolate of the peptide substrate in the presence of bound FPP.
Recombinant Rat FTase was produced
and purified as described (9). Apo-FTase was prepared by dialyzing the
holo-enzyme (>1 mg/ml) for 24 h against 50 mM
Tris-Cl, pH 7.8, 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Buffer 1) and 5 mM EDTA at 4 °C
followed by an additional 24 h dialysis against Buffer 1 containing 50 µM EDTA. After dialysis, the apo-FTase was
concentrated to ~100 µM, flash-frozen, and stored at
70 °C.
Recombinant H-Ras was
purified from a bacterial expression system as described (1, 17).
Residual metals in the H-Ras preparation were removed by dialysis
against Buffer 1 containing 50 µM EDTA and 30 µM guanosine 5-diphosphate at 4 °C for 24 h. Metal ions in the [3H]farnesyl diphosphate
(3H-FPP) (American Radiolabeled Chemicals) were removed by
incubation of the solution with Chelex-100 resin
(Sigma) for 5-10 min. The final preparation of
3H-FPP (20 µM, 8 Ci/mmol) was stored at
18 °C.
FTase was reconstituted with different divalent metals (Zn2+, Co2+, Cd2+, or Ni2+) by incubating the apo-enzyme (150 µg/ml) with 60 µM of the respective metal salt (atomic absorption grade, Aldrich) in Buffer 1 and 50 µM EDTA for 5-10 min on ice before dilution into the assay mixture. For reactions containing dithiothreitol (DTT), the reducing agent (1 mM) was added to the apoenzyme prior to the addition of the metal.
FTase Activity AssaysFTase activity was determined by quantifying the amount of 3H transferred from 3H-FPP into H-Ras as described previously (1, 17). The standard activity was measured with the following components: 8 µM H-Ras, 1 µM 3H-FPP, 0.16 µM FTase, Buffer 1, 50 µM EDTA, and 60 µM of either Zn2+, Co2+, Cd2+, or Ni2+ in the presence or the absence of 5 mM MgCl2 and/or 1 mM DTT. The assay was initiated by the addition of metal-reconstituted FTase and incubated at 30 °C for 1 min. The amount of trichloroacetic acid-precipitable 3H-farnesylated H-Ras was determined by a filter binding assay (1). Under these conditions, the activity of the Zn2+-FTase was 770 nmol/h/mg.
Co2+ Spectra of FTase and FTase ComplexesThe
FPP analogue
{(E,E)-2-[2-oxo-2-[[(3,7,11-trimethyl-2,6,10-docecatrienyl)oxy]amino]ethyl]phosphonic
acid, sodium} (designated compound Ia, see Fig. 1) (18)
and the peptide analogue B581 (designated compound Ib, see
Fig. 1) (19) were purchased from Calbiochem and Bachem,
respectively. The substrate peptide, sequence TKCVIM, was synthesized
on an Applied Biosystems synthesizer and purified as described (9).
Co2+-FTase was prepared by mixing apo-FTase (40-60
µM) with excess CoCl2 (90-120
µM) in Buffer 1, 5 mM MgCl2, and
50 µM EDTA at room temperature. A sample of
Zn2+-FTase was prepared for the reference cuvette by
substituting ZnCl2 in the incubation. The various complexes
were formed by adding a 1:1 stoichiometry of the inhibitor
(Ia or Ib) and/or substrate (FPP or TKCVIM
peptide) to both the sample and reference cuvette. Optical absorption
spectra of Co2+-substituted FTase complexes were recorded
on a Uvikon 9410 UV/VIS double beam spectrophotometer using a 120-µl
cuvette. Each spectrum was measured 2-3 times and then averaged. The
calculated extinction coefficients for each spectrum were reproducible
to within 10%.
Transient Absorption Spectrum
The transient absorbance at 340 nm was measured on a KinTek stopped flow spectrophotometer either by mixing 50 µM Co2+-FTase·FPP binary complex (1:1 stoichiometry of enzyme to FPP) with 10 µM peptide substrate, TKCVIM, to form an active ternary complex or by mixing 50 µM peptide substrate, TKCVIM, with 10 µM Co2+-E·Ia to form an inactive ternary complex in Buffer 1, 5 mM MgCl2, and 50 µM EDTA at 23 ± 1 °C. The absorbance transient was fit to an equation describing either a single first-order exponential (inactive ternary complex) or two consecutive first-order reactions (active ternary complex) (20) by nonlinear regression using the Marquat-Levenberg method provided in the KinTek software package (21).
Miscellaneous ProceduresAll solutions were prepared with
deionized water (18 M) in plasticware presoaked in 5 mM
EDTA followed by deionized water. Protein concentrations were
determined by the Coomassie Blue binding method using a commercial kit
(Bio-Rad).
Previous metal
substitution experiments indicated that Zn2+ and
Cd2+ restore activity to apo-FTase, whereas
Ni2+, Mn2+, Hg2+, and
Co2+ do not (10). Because Co2+ can generally
substitute for Zn2+ in other zinc metalloenzymes (15), we
retested the activity of the Co2+-FTase in an assay with
TCEP substituted for DTT. (DTT catalyzes the formation of
Co3+ from Co2+ in the presence of oxygen while
TCEP reacts more slowly (22, 23).) Using these conditions, the activity
of Co2+-FTase is comparable with that of
Zn2+-FTase (Fig. 2). This restoration of
activity by Co2+ is not due to trace contamination of
Zn2+ in the assay solutions for the following reasons.
First, the high concentration of FTase employed in this reconstitution
experiment (0.16 µM) made it very unlikely that
Zn2+-FTase could form quantitatively in the assay. Second,
the addition of 1 mM DTT to apo-FTase before the addition
of metal had no effect on the activity of Zn2+-FTase but
abolished 75% of the activity in the presence of Co2+
(Fig. 2), and third, the addition of Ni2+, which binds EDTA
with an affinity greater than either Zn2+ or
Co2+ (24), does not restore the activity of apo-FTase (Fig.
2).
Absorption Spectra of Co2+-FTase
Substitution of
Co2+ into the zinc binding site of proteins provides a
useful spectroscopic probe of the composition and geometry of the metal
polyhedron (15, 16). Because the high catalytic activity of
Co2+-FTase indicates that the metal binding site in the
enzyme is not significantly disturbed by this replacement, spectral
information obtained from Co2+-FTase is both structurally
and catalytically relevant. Indeed, the optical absorption spectrum of
Co2+-substituted FTase (Fig. 3A,
solid line) displays characteristic features of the spectra
of many Co2+-substituted zinc enzymes (15, 16). The low
wavelength absorbance (below 450 nm) is assigned to a ligand-metal
charge transfer (LMCT) band indicative of sulfur-to-cobalt charge
transfer resulting from thiolate coordination (16). Furthermore, the
intensity of the absorption shoulder at 320 nm (320 = 1030 M
1 cm
1) is consistent with
one thiolate ligand; the extinction coefficient of LMCT bands are
normally 900-1300 M
1 cm
1 per
cobalt-thiolate bond (25). Consistent with this assignment, an
essential cysteine in the
subunit of FTase, Cys299, has been identified that exhibits the characteristics of a zinc ligand (11). In
addition, the intensity of the ligand field absorption bands that are
observed in the low energy region of the spectrum (
560 = 140 M
1 cm
1;
635 = 100 M
1 cm
1) is indicative of
a pentacoordinate or distorted tetrahedral metal geometry (16). Removal
of the magnesium ion from the solution did not alter the absorption
spectrum (data not shown).
Absorption Spectra of Co2+-FTase Binary Complexes
The addition of FPP to Co2+-FTase to form
the binary complex has little influence on the optical spectrum (Fig.
3A, dotted line), except that the extinction
coefficient of the ligand field bands decreases (560 = 100 M
1 cm
1). This diminution
suggests a slight alteration in the geometry of the metal polyhedron,
perhaps caused by a protein conformational change or interaction of the
pyrophosphate group of FPP with Co2+. Additionally, the
spectrum of Co2+-FTase with a bound FPP analogue (compound
Ia, see Fig. 1) is virtually identical to the spectrum of
E·FPP (Fig. 3, A and B), suggesting that FPP
and Ia bind to the enzyme in a similar manner. Finally, the
shape of the spectrum of Co2+-FTase in the presence of the
peptide substrate TKCVIM is indistinguishable from that of
Co2+-FTase alone, although the extinction coefficients of
both the ligand field and the LMCT bands increase slightly upon the
addition of peptide (<20%; data not shown). The marginal influence of
peptide binding on the absorption spectrum suggests that cobalt does
not coordinate the thiolate of the peptide substrate in the binary complex.
To probe the environment of the metal
binding site in the ternary complex, we used the aforementioned FPP
analogue (compound Ia) that binds to FTase but is not
utilized in catalysis to form an inactive but stable ternary complex
mimic. The spectrum of the
Co2+-FTase·Ia·TKCVIM ternary complex (Fig.
3B) exhibits quite striking differences from the absorption
spectrum of either the E·Ia or E·TKCVIM binary complex.
Most importantly, the intensity of the LMCT band at 320 nm essentially
doubles (320 = 1830 M
1
cm
1), indicating the formation of a second
Co2+-thiolate bond, consistent with coordination of the
thiolate of the peptide substrate with Co2+ in this ternary
complex. Furthermore, the increased intensity of the ligand field
absorption bands at higher wavelengths in the
E·Ia·TKCVIM complex compared with those in the
E·Ia binary complex is also consistent with additional
coordinating thiolates (15, 27, 28, 29). Finally, the increased extinction
coefficient of the d-d transition maximum at 635 nm (
635 = 310 M
1 cm
1) is typical of a
cobalt binding site with tetrahedral geometry (30). The observed
extinction coefficients at both 320 and 640 nm increase linearly with
the concentration of added peptide before saturating at a peptide
concentration equal to the enzyme concentration (Fig. 3C),
providing compelling evidence that (i) the peptide binds
stoichiometrically to the E·Ia complex and (ii) the
increased extinction coefficient observed upon addition of peptide
reflects the spectrum of the E·Ia·TKCVIM complex and
not the absorption of nonspecific Co2+·TKCVIM
complexes.
We also formed the ternary complex using authentic FPP and an analog of
a peptide substrate, B581 (compound Ib, see Fig. 1), that
cannot be used in catalysis. The spectrum of the resultant Co2+-FTase·FPP·Ib ternary complex retains
the significant spectral features of the
Co2+-FTase·Ia·TKCVIM complex described
above, including the increase in the LMCT band (320 = 1900 M
1 cm
1) and changes in the
shape and extinction coefficients of the ligand field absorption bands
(
560 = 150 M
1
cm
1;
610 = 170 M
1 cm
1;
660 = 150 M
1 cm
1) (data not shown).
This similarity suggests that the observed spectral properties are
general features of Co2+-FTase ternary complexes rather
than unique to the inactive E·Ia·TKCVIM complex. Taken
together, these spectral data provide direct evidence that the cysteine
thiol(ate) of the peptide substrate directly coordinates
Co2+ in these ternary complexes.
Because the equilibrium constant for the formation of
farnesylated peptide is very large (26), the reaction of FTase·FPP with one equivalent of the peptide substrate should produce a stoichiometric amount of product bound to FTase. Furthermore, under
these conditions the enzyme should not turnover or release the
farnesylated peptide. We therefore used this method to prepare the
Co2+-FTase·farnesylated-peptide product complex and
recorded its spectrum (Fig. 3A). Examination of the spectrum
of the product complex reveals features that are distinct from those of
either the binary or ternary complexes described above. The LMCT band
in the product complex displays two distinctive shoulders at 320 and
370 nm (320 = 1600 M
1
cm
1 and
370 = 650 M
1 cm
1). However, as observed
in the ternary complexes, the extinction coefficient at 320 nm in the
product complex is significantly greater than that in the
Co2+-FTase·FPP binary complex, indicative of additional
sulfur-cobalt coordination in the product complex. On the other hand,
the shape and extinction coefficient (
560 = 130 M
1 cm
1) of the ligand field
absorption band resembles the Co2+-E·FPP binary complex
rather than the ternary complexes. These spectral changes also increase
linearly upon the addition of limiting TKCVIM to the E·FPP complex
and saturate when one equivalent of the peptide is added (data not
shown), indicating that the spectral changes are due to an interaction
with Co2+-FTase. This is a surprising result because metal
coordination by a thioether in a zinc metalloenzyme has only been
implicated in one previous case, the DNA repair protein Ada (12, 31, 32). However, an interaction between a thioether sulfur and Co2+ has been observed in Co2+-substituted
azurin (33), and ligand-metal charge transfer has been detected between
a thioether sulfur and Co3+ in model compounds (34). The
observed spectral changes in the product complex suggest that the
thioether moiety of the farnesylated peptide product interacts with
Co2+ in the enzyme·product complex.
To verify that the increase in the LMCT band observed in
inactive ternary complexes also occurs in an active ternary complex, we
measured the transient absorbance at 340 nm after mixing excess E·FPP
with TKCVIM (Fig. 4). The transient increase in
absorbance caused by formation of an E·FPP·TKCVIM complex followed
by an exponential decay due to formation of the product complex is
exactly the behavior predicted from the static spectra (Fig. 3,
A and B). These data clearly demonstrate that the
peptide thiol also interacts with the metal ion in the active ternary
complex. A two-exponential fit of these data indicates that the rate
constant for formation of the ternary complex is 60 s1,
consistent with a second-order rate constant of 1 × 106 M
1 s
1, and the
rate constant for product formation is 17 s
1 (Fig. 4). A
single exponential increase was observed when excess TKCVIM was mixed
with E·Ia to form an inactive ternary complex with a
second-order rate constant of 1 × 106
M
1 s
1 (data not shown). Our
data are compatible with previous transient kinetic measurements where
the rate constant of the chemical step and the second-order rate
constant in Zn2+-FTase were determined as >12
s
1 and 2 × 105
M
1 s
1, respectively (26).
In conclusion, these spectral studies clearly indicate that the metal
polyhedron of FTase has either pentacoordinate or distorted tetrahedral
geometry and contains one cysteine ligand. Furthermore, these data
demonstrate that both the peptide substrate (in the ternary complex)
and the farnesylated peptide product bind FTase in such a way that the
Co2+ directly coordinates the sulfur atom. Minimally, this
places the divalent metal ion at the active site of FTase and
demonstrates the presence of an open coordination sphere, most likely
due to the presence of at least one water molecule in the metal
coordination polyhedron. These features strongly argue that the zinc
ion in FTase plays a catalytic rather than a structural role (30). In
fact, these spectral data are completely consistent with the properties
of the metal site in the Ada protein, where the bound zinc activates
the thiol of a cysteine residue for nucleophilic attack on the carbon
of an Sp-methylphosphotriester (12, 31, 32).
Furthermore, catalysis of S-alkylation by Zn2+
coordination of thiol compounds has also been implicated in
cobalamin-independent methionine synthase (35) and several chemical
reactions (36, 37, 38). Therefore, we propose a minimal scheme of the
molecular mechanism of FTase (Fig. 5) in which the zinc
ion in FTase activates the thiol of the peptide substrate for
nucleophilic attack. Studies investigating the rate constant of the
chemical step in metal-substituted FTase should further illuminate the
function of the zinc ion and the catalytic mechanism of FTase.
We thank H.-W. Fu for helpful discussions and John Moomaw and Carolyn Diesing for preparation of FTase and protein substrates. We also thank the Keck Foundation for the support of the Levine Science Research Center at Duke University.