THE JOURNAL OF RIOLOGIC~L CHEM~~Y

Vol. 246, No. 3, Issue of February 10, pp. 772-779, 1971

Printed in U.S.A.

The Mechanism of Aconitase Action

I. PREPARATION, PHYSICAL PROPERTIES OF THE ENZYME, AND ACTIVATION BY IRON(

(Received for publication, August 17, 1970)

JOSEPH J. VILLAFRANCA~ AND ALBERT S. MILDVANS

From The Institute for Cancer Research, Fox Chase, Philadelphia, Pennsylvania 19111

SUMMARY

Pig heart muscle aconitase has been purified to appar- ent homogeneity as judged by polyacrylamide gel electro- phoresis and sedimentation in the ultracentrifuge. The protein has an ~20,~ = 6.16, a molecular weight of 89,000, a Dzo,w value of 6.68 X lo-’ cm2 per set, a frictional ratio, f/f,,, of 1.08, and an isoelectric point of 9.1. The presence of 1.02 f 0.11 moles of iron per 89,000 g of aconitase is revealed by atomic absorption spectroscopy. At 77”K, the electron spin resonance spectrum of aconitase has a signal at g = 4.1 arising from high spin iron(II1). The iron(III) is thought to have a structural rather than a catalytic role since the electron spin resonance spectrum is unchanged upon addition of iron@), cysteine, or citrate. The optical spectrum of aconitase shows a broad nonspecific absorption from 300 to 600 mn at 25”.

Aconitase specifically requires iron for activity and the time-dependent activation by iron(U) is fit by the consecutive combination of 2 iron(H) ions with the enzyme.

The transverse relaxation rate, l/Tz, of aconitase-bound iron(I1l) decreases with increasing temperature and the longitudinal relaxation rate, 1 /Tl, increases with temperature below 22’ and decreases with increasing temperature above 22”, yielding a correlation time TV = 1 x lo-** sec. The inequality of the longitudinal and transverse relaxation rates indicates that protons are rapidly exchanging (>6 x lo5 see-1) between bulk solvent and the coordination sphere of the iron(II1). The molar relaxivity, l/Tp[Fe], for aconitase (5900 M-I se@) at 22’ indicates that aconitase-bound iron(III) is about half as effective as free iron(m) in relaxing the protons of water. From the Solomon-Bloembergen equation and assuming an iron to proton distance of 2.4 f 0.1 A from crystallographic data, it is calculated that 3 f 1 water molecules remain in the inner coordination sphere of aconitase-bound iron(II1).

* This work was supported by National Science Foundation Grant GB-8579, United States Public Health Service Grants AM-13351, CA-06927, and RR-05539 from the National Institutes of Health, and American Cancer Society Grant IN-49, and by an appropriation from the Commonwealth of Pennsylvania.

$ Recipient of a United States Public Health Service Postdoc- toral Fellowship.

5 Established Investigator of the American Heart Association.

Aconitase (citrate (isocitrate) hydrolyase, EC 4.2.1.3) is representative of a class of lyases which are activated by the addition of iron (II) (1). This class includes such enzymes as D-

altronic dehydrase (2), tartrate dehydrase (3), and maleate hydratase (4). A number of authors have suggested that iron(I1) might function in an enzyme-metal-substrate bridge comples 0, 5).

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-hydroxyethylpipera- sine-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 spectro- photometer, 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 triethanol- ammonium-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 reso- nance; ESR, electron spin resonance.

772

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Issue of February 10, 1971 J. J. Villafranca and A. S. Mildvan 773

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 cis- aconitate (9). In most cases no correction for change in ab- sorbance was needed. One unit of enzyme activity was defined as the production of 1.0 pmole of product per min. For homo- geneous 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 pre- pared 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.

RESULTS

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 Tris- citrate 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, con- taining 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 stir- ring to the enzyme solution. After centrifugation at 18,000 X g for 20 min the precipitate was discarded and ammonium sul- fate (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 pipet- ting 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 so- lution was applied to the top of a column, 45 x 1.5 cm, of Seph- adex 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 equil- brating 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 be- tween 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 purifi- cation procedure. Attempts were made to purify aconitase by

z We wish to thank Dr. W. J. Ray for suggesting the liquid nitro- gen method of storage which he has developed to stabilize phos- phoglucomutase.

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774 Mechanism of Acmitase Action. I Vol. 246, No. 3

TABLE I Purification of aconitase from pig heart

The assay used was the rate of formation of isocitrate (8). I I I I I I I

-------

ml I.U./ml I.U. nag/ml I$/ % -fold

Initial extract. . . . . 2850 1.484218 12.5 0.12 100 1 Cm-cellulose chro-

matography.. . . . 200 12.1 2420 3.803.17 57 26 (NH4),SO* frac-

tionation.. . . . . . . . 15 105.0 1570 21.4 4.90 37 40 Sephadex G-200

chromatography&. 6 20.2 121 2.22 9.10 (20)” 76

0 This step was carried out on seven separate aliquots from the preceding step.

b This number represents the yield obtained from the Sephadex G-200 chromatography step calculated from the sum of the seven separate aliquots.

isoelectric focusing but the enzyme consistently migrated into the NaOH as a result of its high p1 value and because the buffer- ing 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 cis- aconitate 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 dis- placement) 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

sRT

D = M(1 - iip) (1)

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 sub- ject 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).

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+.*, I

6 7 8 9 IO PH

FIG. 2. Cellulose acetate electrophoresis of aconitase. Electro- phoresis was conducted in 25 mM potassium phosphate buffer (adjusted to the indicated pH) at 250 volts for 15 min in a Beckman Microzone electrophoresis apparatus. Amido black was used as a protein stain.

1.5000 , I

1.0000 .

0.5000 - __

hC

.oooo L 43.0 43.5 44.0

r’ cm*

FIG. 3. Equilibrium sedimentation of aconitase. The above plot is representative of several determinations made at different protein concentrations (0.49 to 0.98 mg per ml of aconitase dissolved in 50 mM Tris-Cl, pH 7.5) and different rotor speeds (18,000 to 26,000 rpm). Equilibrium was established in each experiment by sedimentation for 24 hours at the desired rotor speed. The temperature was 10”. The ordinate is the logarithm of the fringe displacement, c, in arbitrary units.

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) re- vealed 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 aconi- tase.

Attempts to remove the tightly bound iron of aconitase were unsuccessful. When the enzyme was treated with a 50- fold 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 com- pound (m-phenanthroline) was used. When the same concen- tration 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.

TABLE II

Stoichiometry of iron in aconitase as determined by atomic absorption spectrophotometry and electron paramagnetic resonance

Method of analysis Iron content

Atomic absorption’“. . . . . . 6 Electron paramagnetic reso-

nanceb. . . . . 2

g atoms/mle oconitnse

1.02 f .ll

0.71 f .20

D Aconitase was dissolved in 20 mM Tris-Cl, pH 8.5, containing 0.1 mu EDTA and dialyzed against this buffer overnight. Ali- quots 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).

1.9

1.8

1.7

.6

E = .4 x 5 .3 2 -=l .2

.I

nm

FIG. 4. Optical spectrum of aconitase. A, 1.37 mg per ml of aconitase in 50 mM Tris-Cl, pH 7.5. B, 0.16 mg per ml of aconitase in the same buffer. T = 25”, Light path, 1.0 cm.

Overnight dialysis against o-phenenthrolme or m-phenanthro- line 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 aconi- tase 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).

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776 Mechanism of Aconitase Action. I Vol. 246, No. 3

3 (I value 2

GAUSS ‘i

500 1000 1500 2000 2500 3000 3500 4000

FIG. 5. Difference ESR spectra (derivative of absorption plotted against magnetic field in gauss) of aconitase and Fe (NOs) 3. A, aconitase (19.7 mg per ml) in 50 mM Tris-Cl, pH 7.5, minus base line (Curve C). B, 250 pM Fe(NOa)s in 0.1 M HNOB minus base line (Curve D). C, 50 mM Tris-Cl, pH 7.5. D, 0.1 M HNOa. The ESR spectra were obtained with a Varian E-4 ESR spectrom- eter as described under “Methods”; sweep time, 16 min; time constant 3 set; gain, A and C, 2.5 X 103, B and D, 1.5 X 103; micro- wave power, 200 milliwatts; microwave frequency 9.188 GHz; and T = 77°K. The base lines C and D were subtracted from the spec- tra to yield A and B with the Fabritek 1074 computer.

The molar extinction coefficients at 490 nm for these three pro- teins 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) .

500 1000 1500 2000 2500 GAUSS

FIG. 6. ESR spectra of aconitase in the absence and presence of citrate. 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, con- taining 10 mM potassium citrate. The base line for these spectra, which is given in Fig. 5C from 500 to 2500 gauss, was not sub- tracted. The spectra were otherwise recorded as in Fig. 5 with a gain setting of 2.5 X 103. When 0.16 mM FeClz or 8.8 mM cysteine was added to aconitase, the ESR spectrum remained qualitatively the same as in B. When FeClz and cysteine were both added to aconitase in the above concentrations the electron paramagnetic resonance spectrum remained unchanged.

8000. A

z z:z

E gj

IO00 _

3 8000. B .

ii 6000. 5

G 4000.

2000

t IOOOl

3.1 3.2 3.3 3.4 3.5 3.6 3.7 103/V

FIG. 7. Arrhenius plot of the longitudinal and transverse re- laxation rates of aconitase per mole of iron. A, aconitase (19.5 mg per ml) was dissolved in 50 mM Tris-Cl, pH 7.5. The open squares represent 1/Tip measurements at 24.3 MHz by the pulsed method and the solid squares represent l/Tzp measurements with the Carr-Purcell apparatus. B, aconitase solutions were as above but also contained 12.5 mM potassium citrate. The open circles are ~/TIP measurements and the solid circles represent l/Tzp measurements. The diamagnetic correction was the respective relaxation rates of the buffer alone. The diamagnetic effects of the protein were negligible under these conditions (7). All relaxa- tion rates are normalized by dividing by the concentration of iron(II1).

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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 trans- verse (l/Tap) relaxation rates of the protons of water were studied as a function of temperature. An Arrhenius plot of the longi- tudinal 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 de- creases 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 coordina- tion sphere of the iron (24). The l/TJFe] value for aconitase (5900 M+ see-l) at 22” is much greater than the value for ferre- doxin (~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 coordi- nation 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 average iron to oxygen distance is 1.93 f 0.08 A. This value together with the Fe-O-H bond angle of 124” (assuming axial symmetry of the water molecule with respect to the Fe-O axis) and the O-H distance of 0.96 A (29) yields an average iron to proton distance of 2.45 f 0.08 A.

TIME (mln)

FIG. 8. Time course of activation of pig heart aconitase by iron(I1). Aconitase was activated as described under “Methods” and 10.~1 aliquots were removed for assay by the method of Rose and O’Connell (8) at the times indicated. The data were analyzed according to Equation 5 in the text. T = 25’.

6

0 100 200 300 400 500

TOTAL (Fe) UM

FIG. 9. Plot of the pseudo-first order rate constants against iron(I1) concentration. The pseudo-first order rate constants (k’r = kl[Fe]) obtained from a computer fit of the data in Fig. 8 were plotted as a function of the iron(U) concentration used in each experiment and the second order rate constants calculated from the slopes.

The points of Fig. 8 show that the time dependence of enzyme activation is sigmoidal in character. The curves drawn through the points in Fig. 8 are the theoretical curves derived assuming the mechanism in Equation 4 and values of kl and kz of 100 M-’ see-l and 250 M-I set-l, respectively.

7cD’el MFel E(insctive) ___f E(Fe)(inaotive) - E(Fe)e(aotive)

(4)

The activation process involves consecutive second order re- actions. Equation 5 is the integrated pseudo-first order rate equation for this kinetic scheme which is derived by assuming an excess of iron (30).

IEcactive)l lE(FeX 1 - = ~ = l + (]c, - k*) BIT [UT

(k3e-h(Fe) t-JCle-k(Fe) t) (5)

Equation 5 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

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778 Mechanism of Aconitase Action. I Vol. 246, No. 3

product of a second order rate constant and the concentration of iron(I1) to the first power (VI = kl[Fe]) as shown in Fig. 9. Hence the time course of activation of aconitase by iron(I1) may be interpreted to involve binding in consecutive second order steps to two classes of iron-binding sites which are distinguishable kinetically. These sites may or may not be structurally identi- cal.

DISCUSSION

Aconitase has been purified to apparent homogeneity as judged by electrophoresis on cellulose and polyacrylamide gels and by sedimentation as a symmetrical peak in the ultracentrifuge. The purified enzyme is a globular protein which contains 1 atom of ferric iron as judged by its ESR spectrum. From an exami- nation of the optical spectrum of aconitase, the structural iron(II1) is not present in a heme moiety, nor in a ferredoxin- or rubredoxin-like environment, but rather in an environment similar to conalbumin or transferrin.

The ESR spectrum supports this view since the principal g value for aconitase-bound iron is 4.1 which is similar to conal- bumin and transferrin but not ferrihemoglobin (principal g values of 6 and 2) (31) or ferredoxin (g = 1.94) (32). The similarity of the ESR spectra of aconitase and ferric transferrin suggest that iron is bound in a like manner in these proteins. Moreover, transfer&s from several sources have molecular weights in the range 70,000 to 90,000 and bind iron(II1) at two sites which are identical and noninteracting (33). However, aconitase and transferrin are probably not identical proteins since we find that fresh human serum and plasma (which contains 30 to 60 PM transferrin (34)) shows negligible aconitase activity. Also, serum does not contain aconitase inhibitors since human serum did not inhibit the activity of purified pig heart aconitase.

The longitudinal molar relaxivity of aconitase (5900 ~-l see-l) is much greater than those of spinach ferredoxin (100 M-’ see-l)

(24)) clostridial ferredoxin (3 M-l set-l) , and rubredoxin (600 Mm1 set-1) (35) which have no water ligands, but is very similar to that of transferrin (5500 M-l set-l at 4.2 MHz) which has water ligands (36).

The bound iron(II1) of aconitase does not appear to be directly involved in the catalytic action of the enzyme by the following criteria: (a) the ESR spectrum of aconitase-bound iron is not perturbed by citrate or by the compounds required for activation of the enzyme, namely, iron(I1) and cysteine. (a) the molar relaxivity of the aconitase-bound iron is not lowered by the addition of substrates or cysteine, suggesting that no water ligands are displaced by these compounds, and (c) the enzyme- iron(II1) complex has no detectable effect on the NMR spectrum of citrate or isocitrate. Hence, the bound iron(II1) may serve to stabilize the structure of aconitase. Alternatively, iron(II1) may merely be a stoichiometric tightly bound contaminant. It has not been possible thus far to distinguish among these alter- natives.

The activation of aconitase by iron(I1) and cysteine reveals two kinetically distinct iron-binding steps, each dependent on the concentration of iron to the first power, suggesting that there are two iron(binding sites per 89,000 molecular weight. The kinetic analysis used in this paper does not allow one to state unequivocally whether these two classes of iron-binding sites are structurally similar. Two thermodynamically equivalent nonactivating manganese-binding sites on aconitase have been

detected by ESR, and iron(I1) competes with manganese for these sites.5

The reason for the high specificity of iron(I1) in activating this enzyme is unclear at present but may be related to the unusual kinetics of iron activation. A detailed consideration of these problems will be the subject of a future publication.

AcknowledgmentsWe wish to thank Dr. Joel Rosenblum for performing the sedimentation equilibrium run and for aid in analyzing the data. The authors are also indebted to Miss E. Travaglini of this Institute for the sedimentation velocity determinations and for helpful comments concerning these experiments. We wish to thank Dr. Michael C. Scrutton for helpful discussions concerning the purification of the enzyme and Dr. H. L. Carrel1 for aid with the computer programming.

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Joseph J. Villafranca and Albert S. MildvanPROPERTIES OF THE ENZYME, AND ACTIVATION BY IRON(II)The Mechanism of Aconitase Action: I. PREPARATION, PHYSICAL

1971, 246:772-779.J. Biol. Chem. 

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