COMMUNICATION:
Evidence for a Catalytic Role of Zinc in Protein Farnesyltransferase
SPECTROSCOPY OF Co2+-FARNESYLTRANSFERASE INDICATES METAL COORDINATION OF THE SUBSTRATE THIOLATE*

(Received for publication, October 29, 1996, and in revised form, November 8, 1996)

Chih-Chin Huang Dagger , Patrick J. Casey §par and Carol A. Fierke Dagger par

From the Departments of Dagger  Biochemistry and § Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

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 (epsilon 320 = 1030 M-1 cm-1) consistent with the coordination of one cysteine side chain and also ligand field bands (epsilon 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 (epsilon 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.


INTRODUCTION

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 alpha /beta 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 beta  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.


EXPERIMENTAL PROCEDURES

Preparation of Apo-FTase

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.

Preparation of Metal-free Substrates

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.

Metal Reconstitution Experiments

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 Assays

FTase 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 Complexes

The 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%.


Fig. 1. Structures of FTase substrate analogs used in the formation of ternary complexes. Ia is a FPP analogue (18), and Ib [B581] is a peptide analogue (19). The IC50 for inhibition of farnesyltransferase is 75 nM for Ia and 21 nM for Ib (18, 19).
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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 Procedures

All solutions were prepared with deionized water (18 MOmega ) 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).


RESULTS AND DISCUSSION

Co2+-FTase Is Catalytically Active

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).


Fig. 2. Catalytic activities of metal-substituted FTase. Metal-substituted FTase was prepared, and the catalytic activity of these enzymes was assayed as described under "Experimental Procedures." The relative activities of FTase reconstituted with Zn2+, Co2+, Ni2+, or Cd2+ in the presence (hatched bar) or the absence (solid bar) of 5 mM MgCl2 were assayed in the presence of 8 µM H-ras, 1 µM 3H-FPP, 0.16 µM FTase, Buffer 1, 50 µM EDTA, and 60 µM divalent metal ion at 30 °C. In some cases, 1 mM DTT was preincubated with FTase for 5-10 min before the addition of the metal ion. The activity of Zn2+-FTase in the presence of 1 mM DTT was taken as 100%.
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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 (epsilon 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 beta  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 (epsilon 560 = 140 M-1 cm-1; epsilon 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).


Fig. 3. Optical absorption spectra of Co2+-FTase and its binary and ternary complexes. Apo-FTase was prepared as described under "Experimental Procedures." Co2+-FTase was prepared by incubation of apo-FTase (46-62 µM) with excess CoCl2 (90-120 µM) in Buffer 1, 5 mM MgCl2, and 50 µM EDTA at room temperature. The reference cuvette contained an identical sample, except that ZnCl2 was substituted for CoCl2. A, absorption spectrum of Co2+-FTase (solid line); Co2+-E·FPP complex (dotted line) formed by the addition of 70 µM FPP; and Co2+-E·product complex (dashed line) formed by the addition of 91 µM TKCVIM to the Co2+-E·FPP complex. B, absorption spectra of Co2+-E·Ia complex (dotted line) formed by the addition of 70 µM Ia to Co2+-FTase and the ternary Co2+-FTase·Ia·TKCVIM complex formed by the addition of either 22 µM (dashed line) or 110 µM (solid line) of TKCVIM to FTase·Ia. C, influence of the addition of increasing concentrations of TKCVIM peptide on the observed extinction coefficient at 320 (closed circle) and 640 nm (open circle) as the peptide bound to Co2+-FTase·Ia to form the E·Ia·TKCVIM ternary complex. Saturation occurs when the concentration of added peptide equals the concentration of Co2+-FTase·Ia.
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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 (epsilon 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.

Absorption Spectra of Ternary Complexes of Co2+-FTase

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 (epsilon 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 (epsilon 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 (epsilon 320 = 1900 M-1 cm-1) and changes in the shape and extinction coefficients of the ligand field absorption bands (epsilon 560 = 150 M-1 cm-1; epsilon 610 = 170 M-1 cm-1; epsilon 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.

Absorption Spectrum of the Co2+-FTase Product Complex

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 (epsilon 320 = 1600 M-1 cm-1 and epsilon 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 (epsilon 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.

Transient Absorbance of Active and Inactive Ternary 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 s-1, 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).


Fig. 4. Transient absorbance of the Co2+-FTase·FPP·TKCVIM active ternary complex. A transient increase in absorbance at 340 nm was measured in a KinTek stopped flow spectrophotometer after mixing 50 µM Co2+-FTase·FPP complex with 10 µM TKCVIM to form an active ternary complex. The absorbance then decreases due to the formation of the product complex. The data were fit with an equation describing two consecutive first-order reactions (20) with the following results: k1 = 17 ± 3 s-1, amplitude = 0.029 ± 0.002; and k2 = 60 ± 10 s-1, amplitude = 0.037 ± 0.002.
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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.


Fig. 5. A proposed mechanism for FTase. In the absence of either substrate, the bound metal in FTase coordinates the thiolate of one cysteine residue with either a pentacoordinate or distorted tetrahedral geometry. In the ternary complex, the peptide thiol binds to the metal and is activated to attack the carbon of farnesylpyrophosphate as a nucleophile. In the product complex, the sulfur of the farnesylated peptide remains coordinated with the metal. R, farnesyl group; FPP, farnesyl pyrophosphate; CVIM, tetrapeptide Cys-Val-Ile-Met; E, FTase.
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FOOTNOTES

*   This work was supported by National Institutes of Health Grants GM40602 (to C. A. F.) and GM46372 (to P. J. C.) and by funds from the Council for Tobacco Research (to P. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Established Investigators of the American Heart Association.
par    To whom correspondence should be addressed: Dept. of Biochemistry, Box 3711, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-2557; Fax: 919-684-8885.
1    The abbreviations used are: FTase, protein farnesyltransferase; FPP, farnesyl pyrophosphate; 3H-FPP, tritium-labeled farnesyl pyrophosphate; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; DTT, dithiothreitol; LMCT, ligand-metal charge transfer; TKCVIM, hexapeptide Thr-Lys-Cys-Val-Ile-Met.

Acknowledgments

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.


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