The Mechanism of Aconitase Action

When a number of metal ions were tested as activators over a lOO-fold range of metal ion concentration (0.01 to 1.0 mM), aconitase was specifically activated by Fe(II). Manganese(I1) does not activate aconitase but binds to the enzyme, as shown indirectly by inhibition of the enzyme activation by Fe(I1) and directly by electron paramagnetic resonance. A titration of the enzyme with Mn(I1) may be fit most simply by assuming two “tight” binding sites for Mn(I1) with a dissociation constant, Kdissp of 47 f 3 PM and 5 to 7 “weak” binding sites (Kdiss = 620 f 80 PM). Interaction among these sites is indicated by the effect of bound Mn(I1) on the longitudinal relaxation rate (l/TIP) of water protons. Iron(I1) competes with Mn(II) for the tight binding sites, yielding a dissociation constant for aconitase-Fe(I1) of 16 =t 6 PM. The binary aconitase-Fe(I1) complex and the ternary aconitase-Fe(II)-citrate complex are paramagnetic, as determined by l/Tl, of water protons, but less so than unbound Fe(II), as determined by magnetic susceptibility. In the catalytically inactive Mn-aconitase complex, a 3-fold enhancement of the effect of Mn on l/TIP of water protons is observed. Citrate, isocitrate, and cis-aconitate halve this enhancement, suggesting the formation of ternary enzymemetal-substrate (E-&f-S) bridge complexes. The dissociation constants (K3) of these ternary Mn(I1) complexes agree with the respective Km values of citrate and isocitrate, but not of cis-aconitate using the Fe(I1) enzyme. Citrate in proportion to its concentration raises the K3 of E-manganeseisocitrate, consistent with competition of the two substrates for the same Mn(I1) site on the enzyme.

Aconitase was previously prepared from pig heart to about 75% purity (6). Since this enzyme was only partially pure and unstable, a study of its physical properties was impossible. The present paper describes a new purification of aconitase which gives an enzyme at least 95% pure and which was found to contain 1 atom of iron(II1).
The physical properties of this enzyme and the kinetics of its activation by iron(I1) are here investigated. EXPERIMENTAL PROCEDURE 1Materials Citric acid, L-cysteine hydrochloride, N-2-hydroxyethylpiperasine-N'-2-ethanesulfonic acid, triethanolamine hydrochloride (heavy metals: _< 1 ppm) and isocitric dehydrogenase (Grade A) were products of Calbiochem. Mann enzyme grade Tris and (NH4)2S04 were used when indicated. Monopotassium threo-D~( +)-isocitric acid was purchased from Sigma and Whatman CM52-cellulose from H. Reeve Angel. FeCl2 and Fe(NH4S0& were Baker analyzed reagent grade. Deionized water was used throughout. Concentrated buffers and reagents were checked for paramagnetic contaminants by measuring the longitudinal relaxation rate, l/T1, of the protons of water in these solutions by pulsed NMRl at 24.3 MHz (7).

Methods
Enzyme AssaysAconitase was routinely assayed under the conditions of Rose and O'Connell (8) by following the production of NADPH at 340 nm on a Gilford model 240 recording spectrophotometer, For kinetic studies another assay was used based on the ultraviolet absorption of cis-aconitate at 240 nm. At 25", assay solutions contained the following: 20 mM triethanolammonium-Cl (pH 7.5), 5 mivr potassium D,(+)-isocitrate (or citrate) neutralized to pH 7.5, and enzyme in a final volume of 1.0 ml. Absorbance changes were measured with a Gilford model 240 recording spectrophotometer with l.O-cm path length 1 The abbreviations used are: NMR, nuclear magnetic resonance; ESR, electron spin resonance. cells. When ferrous activated aconitase was assayed by this method controls were run without enzyme to ensure that the absorption measured was due to enzymatic production of cisaconitate (9). In most cases no correction for change in absorbance was needed. One unit of enzyme activity was defined as the production of 1.0 pmole of product per min. For homogeneous aconitase, the E:FO was determined to be 13.7 based on the Lowry method (10) with the protein samples prepared by the Schneider procedure (11) with bovine serum albumin as a standard.

Analytical
Ultracentrifugation-A Beckman-Spinco model E analytical ultracentrifuge equipped with ultraviolet absorption optics was used for the sedimentation velocity determination. A model E analytical ultracentrifuge equipped with Rayleigh optics was used for the sedimentation equilibrium experiments.
Iron Analysis-This was performed by atomic absorption with the Techtron atomic absorption spectrophotometer.
Optical Spectra-Spectra of the purified enzyme were recorded on a Cary model 15 recording spectrophotometer.

Magnetic
Resonance Techniques-The longitudinal relaxation rate, l/Tl, and the transverse relaxation rate, l/T2 of the protons ot water were measured in an NMR Specialties PSGOW pulsed NMR spectrometer at 24.3 MHz (7).
The relaxation rates of the carbon bound protons of the substrates were carried out in 99 % DzO with a Varian HA-loo-15 NMR spectrometer as previously described (12).
Iron(III) ESR measurements were made at 77" K with the Varian E-246 quartz Dewar attachment for the Varian E-4 ESR spectrometer.

Kinefics
of Activafion by Iron.(Aconitase was activated by ferrous ion at 25" in a solution containing 40 rnM sodium N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (pH 7.5), 10 mM cysteine (pH 7.5), 25 to 500 pM Fe(NH$O&, and aconitase (added last) in a final volume of 0.1 ml. Aliquots, 5 to 10 ~1, were removed at various times and tested for activity by the assays described above. Solutions of iron(I1) salts were prepared in deionized water which was deaerated by bubbling Nz through it for 5 min. Ferrous ammonium sulfate was added after cysteine in the above solution to minimize the oxidation of iron(I1) to iron(II1) by oxygen dissolved in the buffer.

Preparation of Enzyme
Unless otherwise stated, all manipulations in the following procedure were at O-4'. The enzyme was assayed at each step after activation by iron(I1) as described under "Methods." Step 1: Extraction-Extraction was carried out in a manner similar to that previously reported by Morrison (6) but with the following exceptions: 1 kg of fresh defatted pig heart was cut into l-inch cubes and blended with 3 liters of 4 mu potassium citrate, 0.05 mu EDTA, 10 mu mercaptoethanol buffer which had been adjusted to pH 5.0 with KOH and 400 ml of CHCI, for 3 min. The product was centrifuged and the supernatant was filtered through cheesecloth (2850 ml). The pH was adjusted to'6.1 with solid Tris.
Step 2: Carboxymethyl Cellulose Column Chromatography-About 500 ml of a thick slurry of Whatman CM52-cellulose equilibrated with 2 mu Tris-citrate buffer, pH 6.1, containing 0.15 mu EDTA were added to the above supernatant and the suspension was stirred for 15 min. The suspension was allowed to settle and the supernatant assayed for enzyme activity. hlore Whatman CM52-cellulose slurry was added with stirring to absorb the aconitase (usually no more than an additional 100 ml). After this second addition of carboxymethyl cellulose usually no more than 20% of the total aconitase enzyme units from Step 1 were in the supernatant. The suspension was centrifuged and the supernatant fluid was discarded. The Whatman CM52-cellulose paste was suspended with 500 ml of 2 InM Tris-citrate buffer, pH 6.1, containing 0.15 mM EDTA, and a Whatman CM52-cellulose column, 60 x 2.5 cm, was poured. The column was washed with the same buffer until the Atso of the effluent matched that of the buffer. The aconitase was then eluted with a linear gradient consisting of 450 ml of 2 mu Triscitrate buffer, pH 6.1, containing 0.15 mM EDTA, in the mixing chamber and 450 ml of 30 mM Tris-citrate buffer, pH 6.5, containing 0.15 mM EDTA, in the reservoir. Fractions of 10 ml were collected and the enzyme was eluted after about 850 ml of effluent volume. The fractions with maximum aconitase ac.tivity were pooled (200 ml).
Step S: Ammonium Sulfate Fractionation-Solid ammonium sulfate (0.415 g per ml of protein solution) was added with stirring to the enzyme solution. After centrifugation at 18,000 X g for 20 min the precipitate was discarded and ammonium sulfate (0.19 g per ml) was added to the supernatant fluid. The suspension was centrifuged as described above and the resultant precipitate was dissolved in 2 mM Tris-citrate buffer, pH 6.5, containing 0.15 InM EDTA, 0.05 mM dithiothreithol (15-ml final volume). The enzyme was stored at this stage in liquid nitrogen.2 The aconitase solution was quickly frozen by pipetting it dropwise into liquid nitrogen and the frozen enzyme pellets were stored in a plastic bottle under liquid nitrogen. The enzyme had not lost activity after 8 months of storage.
Step 4: Sephadex G-200 Column Chromatography-Twenty-five milligrams of protein from Step 3 were quickly thawed, dialyzed, and concentrated in a Sartorius protein concentrator equipped with collodion membrane filters. The dialyzing buffer (20 mM Tris, 1 mM citrate buffer, pH 8.5, containing 0.1 mu EDTA, 0.1 mM dithiothreitol) was changed four times while the protein was concentrating and additional buffer was added to the protein solution to avoid precipitation of the protein. The final protein concentration achieved was -25 mg per ml. This enzyme solution was applied to the top of a column, 45 x 1.5 cm, of Sephadex G-200 which had been washed with 0.1 M sodium EDTA, pH 7.0, and then equilibrated with the same buffer used above for the protein dialysis. The enzyme was eluted with the equilbrating buffer at a flow rate of 5 ml per hour. Fractions of 1 ml were collected and the fractions with the highest specific activity were pooled. The enzyme eluted as a symmetrical protein peak. Pooled enzyme was stored frozen in liquid nitrogen as described above. The results of the purification are given in Table I.
Purified aconitase yielded a single band on disc electrophoresis at pH 9.5 ( Fig. 1) and on cellulose acetate electrophoresis from pH 6.5 to 9.8. The p1 of the enzyme was estimated to be between 9.0 and 9.3 as judged from its behavior on cellulose acetate (Fig. 2). The basic nature of the protein is evident by its binding to carboxymethyl cellulose at pH 6.1 during the purification procedure. Attempts were made to purify aconitase by Mechanism of Acmitase Action. I Vol. 246, No. 3 The assay used was the rate of formation of isocitrate (8). I  I  I  I  I  I  I   ------ isoelectric focusing but the enzyme consistently migrated into the NaOH as a result of its high p1 value and because the buffering capacity of ampholites is low in the pH range 9 to 10. The purified enzyme ( Step 4) had about 0.1% of the activity that was obtainable upon activation by iron(I1) and cysteine and the enzyme was stored in liquid nitrogen in the unactivated state. 2 The purified enzyme, when fully activated, had a turnover number per mole of enzyme of 13.5 see-1 for isocitrate formation from citrate, and 122 set-l for isocitrate formation from cisaconitate under the assay conditions described above.

Physical Properties of Enzyme
Analytical Ultracentraj~ation-Examination of aconitase by sedimentation velocity experiments showed a single symmetrical peak. Ten to 25 ~1 of a 3.3 mg per ml solution of aconitase (from Step 4) were layered on 1.0 M NaCl according to the method of Vinograd et al. (15). With ultraviolet absorption optics the experiment was performed at a nominal speed of 59,780 rpm at 21.2' and gave an szo,u, of 6.16. Sedimentation equilibrium experiments were conducted by the method of Yphantis (16) with 0.49 to 0.98 mg per ml of enzyme dissolved in 50 mu Tris-Cl, pH 7.5. At all protein concentrations and rotor speeds (18,000 to 26,000 rpm), the plot of In (fringe displacement) against r2 (Fig. 3) was linear, indicating that the protein was homogeneous and that the solutions did not deviate significantly from ideality. The molecular weight calculated from these determinations was 89,000.
The diffusion coefficient was calculated from the molecular weight, sedimentation coefficient, and an assumed partial specific volume-(u = 0.749) by the use of the Svedberg equation where R is the gas constant and p is the solvent density. A value of D20,(o was calculated to be 6.68 x lo-1 cm2 per sec.
The frictional ratio, j/jo, was calculated with the diffusion coefhcient, molecular weight, and viscosity of the solution from the expression FIG. 1. Polyacrylamide disc gels of aconitase at two stages of purification.
Electrophoresis was performed on acrylamide gels at pH 9.5 as previously described (13) with Amido black used as a protein stain.
The gel on the right represents electrophoresis of an enzyme sample from Step 3 of the purification.
An enzyme sample from Step 4 (Sephadex G-200 chromatography) was subject to electrophoresis under the same conditions described above and was sliced in half before protein staining.
One-half was stained with Amido black and is the gel on the left. The other half was incubated at 37" with citrate, and the isocitrate produced by the action of aconitase was detected by a coupled assay with isocitrate dehydrogenase, TPN, phenasine methosulfate, and nitro-blue tetrazolium (14). The half of the gel stained in this manner is in the middle.
where L is Boltzman's constant and N is Avogadro's number. The frictional ratio equaled 1.08, suggesting that aconitase behaves in the ultracentrifuge as a globular protein with an axial ratio between 1.0 and 2.6 (17). Iron Content-The purified enzyme was brown in color and was therefore analyzed for its iron content by atomic absorption spectroscopy.
The results of an iron analysis (Table II) revealed that iron was present in high enough concentration to be considered as a stoichiometric component of aconitase.
It is evident that iron is either tightly complexed by aconitase or bound at a site inaccessible to EDTA throughout the purification procedure.
The iron content determined by atomic absorption is 1.02 f 0.11 moles of iron per 89,000 g of aconitase.
The ESR spectrum of aconitase at 77" K (see optical and ESR spectrum of aconitase section) was doubly integrated with the Fabritek 1074 instrument computer and the area under the absorption spectrum when compared with a solution of known iron(II.1) concentration gave 0.71 f 0.20 mole of iron per mole of aconitase.
Attempts to remove the tightly bound iron of aconitase were unsuccessful.
When the enzyme was treated with a 50fold excess (-7 X 10-' M) of metal chelator (o-phenanthroline, cycr-bipyridyl, 4,5-dihydroxy-m-benzenedisulfonic acid) at 0 or 25" at pH 7.5 for 10 min to 2 hours, a loss of enzymatic activity resulted, that could not be regained by iron(I1) activation. Activity was similarly lost when a nonchelating aromatic compound (m-phenanthroline) was used. When the same concentration of EDTA, ethylenediamine, or diethylenetriamine were used in such experiments no inactivation was observed since the enzyme could be activated by iron(I1) to the same extent as in the absence of metal chelators. Aliquots of the enzyme (6.2 to ll.Omg per ml) were analyzed for metal content with a Techtron AA-4 atomic absorption spectrometer as described under "Methods." The enzyme and the dialysis buffer were analyzed with an iron standard run before and after each sample.
The metal content was calculated from the increment in the absorbance obtained with each sample as compared with the iron standard.
The iron content of the dialysis buffer was 0.10 pg per ml and was subtracted from each enzyme determination.
b Average of the values obtained by double integration of the electron paramagnetic resonance spectra (see Fig. 5) from two samples of aconitase (7.8 and 19.7 mg of protein per ml). Overnight dialysis against o-phenenthrolme or m-phenanthroline under the above conditions caused irreversible inactivation while dialysis against EDTA had no effect on the activity or iron content.

Optical and Electron Paramagnetic
Resonance Spectra of Aconitase Optical Spectrum of Aconitase-The spectrum of purified aconitase measured from 240 to 600 nm is shown in Fig. 4. The absence of a strongly absorbing region in the visible portion of the spectrum near 412 nm appears to rule out the presence of heme-bound iron in the enzyme. The broad absorption band between 320 and 600 nm exhibited by aconitase does not have the absorption peaks present in other iron-containing proteins such as rubredoxin (380 nm, 490 nm) (X3), ferredoxin, and adrenodoxin (418 nm, 468 nm, 5,15 nm) (19). The optical spectrum of ferric transferrin has a broad absorption band from 320 to 600 nm with a small absorption peak at 470 nm (20).  The molar extinction coefficients at 490 nm for these three proteins are: -3500 M-~ cm-l for aconitase, ~2500 M-' cm+ for transfer& and 9000 M+ cm+ for rubredoxin. Qualitatively, aconitase resembles transferrin more than rubredoxin in the optical region from 320 to 600 nm.
ESR Spectrum of Aconitase-The ESR spectrum of aconitase at 77" K has a strong signal at g = 4.1 (Fig. 5). This resonance has been described by Blumberg (21) as arising from high spin iron(II1) in a crystal field of low symmetry. Ferric transferrin and ferric conalbumin show the same ESR spectrum as aconitase with the main deflection around g = 4.1 (20). Several low molecular weight complexes of iron(II1) exhibit the same type of ESR spectra. Certain of these complexes have a broad resonance line at g = 2 (22) which is also present with aconitase. The additional lines in Fig. 5 at g = 7.3, 5.4, and 4.9 are of unknown origin and their assignments have not been made. Resonance lines at g = 8.8 to 8.9 have been found with ferric transferrin and conalbumin and a shoulder at g = 9.7 has been found in inorganic complexes of iron(II1).
The geometry of the iron giving rise to the g = 4.1 signal could arise from the zero field state of the Y&/Z ion being ellipsoidal in nature. This state might have anisotropic behavior which would explain the g = 7.3, 5.4, and 4.9 signals.
The ESR spectrum of aconitase bound iron was not altered by citrate (Fig. 6) or by the compounds needed to activate the enzyme (iron(I1) or cysteine) . A, aconitase (19.7 mg per ml) in 50 rnM Tris-Cl, pH 7.5. B, aconitase (19.7 mg per ml in 50 mM Tris-Cl, pH 7.5, containing 10 mM potassium citrate. The base line for these spectra, which is given in Fig. 5C from 500 to 2500 gauss, was not subtracted.
The spectra were otherwise recorded as in Fig. 5

Effect of Aconitase on Relaxation
Rates of Protons of Water and Substrate-To determine the accessibility of water to the iron(II1) bound tightly to aconitase the longitudinal (l/T1p) and transverse (l/Tap) relaxation rates of the protons of water were studied as a function of temperature.
An Arrhenius plot of the longitudinal and transverse molar relaxivities (1 /T&Fe] and l/T&Fe]) is given in Fig. 7. The transverse relaxation rate decreases with increasing temperature and the longitudinal relaxation rate increases with temperatures below 22" and decreases with increasing temperatures above 22'. Such findings indicate that protons are rapidly exchanging between the bulk solvent and the coordination sphere of the metal (23). This behavior is unlike that for ferredoxin and adrenodoxin where the longitudinal and transverse relaxation rates were equal and much lower over this temperature range, suggesting that the relaxation takes place by an outer sphere process with no exchange of protons between the solvent and the first coordination sphere of the iron (24). The l/TJFe] value for aconitase (5900 M+ see-l) at 22" is much greater than the value for ferredoxin (~100 M-' seP) and metmyoglobin (-1,000 M-l set-l) but not as large as that of iron(II1) in solution (~11,500 M-l set-1). Since iron(II1) in solution coordinates six water ligands, the fact that aconitase-bound iron is half as effective as free iron in relaxing the protons of water suggests that the iron in aconitase may have retained three water ligands. Since the relaxation process is in the region of rapid exchange it can be seen that l/Tlp[Fe] = q/T&H201 where Q is the coordination number for water and TIM is the relaxation rate of a coordinated ligand (23). From the data q/TIM is 654,900 set-l at 22". An estimate of ~~ was made from the dipolar term of the Solomon-Bloembergen equation (Equation 3) which predicts a maximum in q/TIM when w~'+~Z = 1.3 With the value of 1.0 x 10'1 set-l for the electron resonance frequency (w,) at 24.3 MHz, a value of 1.0 X lo-l1 set was obtained for 7C. Substituting these values into Equation 3 and assuming a distance, r, from the iron to the (3) protons of water of 2.45 f 0.08 A from crystallographic data4 yields a value of q = 3.4 f 1.0 water molecules in the coordination sphere of aconitase-bound iron.
No interaction of cysteine (10 mM) or of the substrates citrate (20 mu), isocitrate (20 mM), or cis-aconitate (15 mu) with the bound iron(II1) was detected by the relaxation rates of water at 24.3 MHz. In addition, no effect of the enzyme-iron(II1) complex (0.2 InM) was detected on the longitudinal or transverse relaxation rates of the protons of citrate (60 mu) or isocitrate (80 mM) by continuous wave NMR at 100 MHz.

Kinetics of Activation of Aconitase by Iron
Aconitase was activated by iron(I1) and cysteine and the activated enByrne was assayed as described under "Methods." 3 The small hyperfine contribution to TIM has been neglected. An alternative possibility of a maximum when ~~27~2 = 1 is ruled out in the case of iron(III), since this would yield a value for r5 (6.6 X 10-9 set) much larger than the electron spin relaxation time of iron(II1) (TV = 5.1 X l(r"sec) (25,26). 4 From x-ray crystallographic studies of iron(II1) compounds (27,28) The activation process involves consecutive second order reactions. Equation 5 is the integrated pseudo-first order rate equation for this kinetic scheme which is derived by assuming an excess of iron (30). shows the time course of activation of aconitase. A computer program was used to generate theoretical curves for various combinations of pseudo-first order rate constants and these curves were used to fit the data in Fig. 8. A good fit was obtained in all cases showing consistency between the assumed mechanism of activation of aconitase and the data. The pseudo-first order rate constants (k'r) which were used to generate the theoretical curves were shown to consist of the