ISOCITRITASE: ENZYME PROPERTIES AND REACTION EQUILIBRIUM* A new aldol reaction has ...

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ISOCITRITASE: ENZYME PROPERTIES
AND REACTION EQUILIBRIUM*
BY ROBERTS A. SMITHt AND I. C. GUNSALUS
(Departments of Bacteriology and Chemistry, University of Illinois, Urbana, Illinois)
(Received for publication, April 25, 1957)
A new aldol reaction has been described (1, 2) in which L.( +)-isocitric
acid (3) is reversibly cleaved to glyoxylic and succinic acids by the enzyme
“isocitritase” (4, 5). This enzyme has been found in a wide variety of
aerobic and facultative bacteria (6) and in yeasts and other fungi (4),
and the growth conditions which favor optimal enzyme formation have
been shown to be aerobic with organic acids as the energy source (6).
Growth of a facultative organism in the presence of glucose completely
repressed isocitritase formation, whereas the aerobic organisms produced
less, but measurable amounts, of the enzyme (6). Earlier publications
(1, 7) from this laboratory reported requirements for a divalent metal
and a sulfhydryl compound, cysteine or GSH,l for maximal enzymatic
activity of a partially purified isocitritase.
The reversibility of the isocitritase reaction was first indicated (8) by
experiments with a crude extract of Pseudomonasa eruginosa, strain 9027,
and was later measured both by coupling to isocitric dehydrogenase and
by exchange experiments with C”-labeled succinate (7). The present
paper concerns the properties and partial purification of isocitritase and
defines the stoichiometry, equilibrium, and energetics of the reaction
catalyzed by this enzyme.
Materials and Methods
Bacteriological-P. aeruginosa, strain 9027, was grown at 30” in a medium
containing, in percentage units, sodium acetate 0.5, NH4HzP04 0.3,
monosodium glutamate 0.1, MgSO4.7HzO 0.1, FeS04*7Hz0 0.0005, and
yeast extract (Difco) 0.01. 14 liters of medium were autoclaved in a 5
gallon Pyrex carboy, cooled, and inoculated in the same medium with 1
* Supported in part by the Atomic Energy Commission and the Office of Naval
Research.
t Du Pont Predoctoral Fellow in Biochemistry, 1954-55, Departments of Bacteri-
ology and Chemistry, University of Illinois.
1 The abbreviations used throughout this paper include GSH, glutathione; TPN+,
triphosphopyridine nucleotide; DiNPH, 2,4-dinitrophenylhydrazine or 2,4-dinitro-
phenylhydrazone; Tris, tris(hydroxymethyl)aminomethane; Versene, ethylenedi-
aminetetraacetic acid; CoA, coenzyme A; TCA, trichloroacetic acid; PS, protamine
solution; GS, gel solution.
305
This is an Open Access article under the CC BY license.
306 ISOCITRITASE
liter of a culture shaken for 16 hours. The 5 gallon culture, when grown
in a laboratory fermenter with continuous aeration (1 volume of air per
minute) and agitation, reached maximal growth in about 7 hours. The
cells were harvested with a Sharples centrifuge and washed once with 0.02
M potassium phosphate buffer, pH 7.0; yield, 6 gm. of wet cells per liter.
The cells were suspended to a level of 250 mg. of wet weight per ml.
of 0.02 M phosphate buffer, pH 7.0, containing 0.5 mg. per ml. of GSH and
were disintegrated by treatment with a 10 kc. Raytheon oscillator for
20 minutes. The cell debris was removed by centrifugation at 32,000 X
g for 1 hour in a type SS-1 Servall centrifuge in a cold room at 5”, and the
supernatant fluid (extract) was used for enzyme fractionations.
Preparations; Chemical-cis-Aconitic anhydride was prepared from
trans-aconitic acid by the method of Malachowski and Maslowski (9).
r,,(+)-Isocitric acid was recovered from dried leaves of Bryophyllum
cwnatum2 as described by Vickery (10) or from fresh Sedum spectdde
leaves and stalks? by alcoholic extraction as follows: Each kilo of fresh
tissue was homogenized batchwise in a Waring blendor with 300 ml. of
50 per cent EtOH and the filtrate was collected through coarse paper.
The residue was pressed in a Carver press, reextracted with 300 ml. of
50 per cent EtOH, and stored overnight at 5’, and the filtrate was again
collected. The combined filtrates were concentrated in vacw, at a tempera-
ture below 40” to yield 2 liters of dark brown syrup. An equal volume of
alcohol was added and the precipitate which formed collected by filtration.
This pectin-containing precipitate was further dehydrated by trituration
with alcohol followed by ether and was finally dried in vacw, at room tem-
perature over CaSOd (Drierite). The product contained about 25 per
cent by weight L,( +)-isocitric acid according to the isocitric dehydrogenase
assay (ll), and treatment of 100 gm. of this material by Vickery’s pro-
cedure (10) yielded 9 to 10 gm. of crystalline L.(-)-dimethylisocitric
la&one (45 per cent yield). The crystalline la&one melted at 105-106”
and showed a rotation [ali -65”. After saponification with KOH, it as-
sayed 100 per cent n,,(+)-isocitrate with isocitric dehydrogenase (11).
Sodium glyoxylate monohydrate was prepared by hydrolysis of di-
bromoacetic acid which was synthesized according to the procedure of
Genvresse (12). 40 ml. aliquots of a 10 per cent aqueous solution of di-
bromoacetic acid were sealed in 70 ml. Carius tubes and heated for 14
hours at 135”. After cooling, the tubes were opened, the contents rinsed
into an evaporating dish, and excess water and HBr removed by evapora-
tion at 110’ on a sand bath until a slight tinge of yellow appeared. This
*We are indebted to the Botany Department of the University of Illinois for a
supply of Btyophyllum crenatum plants.
8 We wish to thank Dr. W. A. Wood for the alcoholic extract of Sedum epectabile.
R. A. SMITH AND I. C. GUNSALUS 307
solution was transferred to a vacuum desiccator and allowed to stand over
PzOa and moist NaOH at room temperature until reduced to a viscous
syrup. The syrup was dissolved in a minimum of water and neutralized
with NaOH, and the sodium glyoxylate monohydrate was crystallized
by the procedure of Metzler et at. (13) (72 per cent yield). The product
was dried in vacua over PzOa and found to assay 100 per cent sodium glyoxy-
late monohydrate by the calorimetric procedure of Friedemann and Haugen
(14). As a standard, a sample was used of crystalline 2,4-dinitrophenylhy-
drazone (DiNPH3) of glyoxylic acid which had been recrystallized twice
from’ 85 per cent methanol (melting point 190”).
Assays; Chemical-Glyoxylate was assayed as its DiNPH by the method
of Friedemann and Haugen (14) except that no solvent extraction was used,
TABLE I
Enzymatic Formation of Glyoxylate from L,(+)-Zsocitrate
-
Components
Complete. 3.55
Minus n.(+)-isocitrate. _. 0
“ cysteine. 0.1
‘I
MgClz 0.1
“
isocitritase.. 0
The complete reaction mixture contained, in micromoles per 1.5 ml.: Tris buffer,
pH 7.6, 100; n.(+)-isocitrate, 5; cysteine hydrochloride, 2; MgClz, 3; and enzyme
(ammonium sulfate III, Table II = 0.11 mg. of protein). Incubation, 10 minutes
at 30” under Nz.
since other keto acids were either absent or were detectable only in trace
amounts by the chromatographic procedure of Cavallini et al. (15).
Radioactivity was measured after wet oxidation of the respective acids
to BaC40a by the persulfate method of Osburn and Werkman (16).
Succinic and isocitric acids were separated on a Dowex 1 column according
to the procedure of Busch et al. (17) after conversion of the glyoxylic acid
to its DiNPH. Glyoxylic acid DiNPH did not migrate and was collected
from the top of the column by extraction with 10 per cent Na2C03 after
the isocitric and succinic acids had been recovered separately.
Assays; Enzymatic-+,.( +) -1socitric acid was assayed by TPN+ reduc-
tion with isocitric dehydrogenase obtained from pig heart according to
Ochoa (11).
Isocitritase activity was measured in an assay system which contained
in 1.5 ml. (13 by 100 mm. test tubes) the following reactants in micro-
moles: Tris buffer, pH 7.6, 190; MgC12 3; cysteine-hydrochloride (un-
308 ISOCITRITASE
neutralized) 2; enzyme and substrate 5. After 10 minutes incubation at
30”, the reaction was stopped by addition of 0.1 ml. of 80 per cent TCA.
For analysis, the mixture was centrifuged to remove protein and an aliquot
containing 0.1 to 1.5 pmoles of glyoxylate was diluted to 1 ml. and added
to 1 ml. of 0.1 per cent DiNPH in 2 N HCl in an 18 mm. calorimeter tube.
After 5 minutes at room temperature, 2 ml. of 95 per cent EtOH, 1 ml.
of HZO, and 5 ml. of 1.5 N NaOH were added and the color intensity was
read in an Evelyn calorimeter with the 540 rnp filter 3 minutes after addi-
tion of the alkali; with 10 ml. volume in an 18 mm. calorimeter tube;
micromoles per sample = 1.07 X absorbance.
1 unit of isocitritaee was defined as that amount of enzyme required to
form 1 rmole of glyoxylate in 10 minutes at 30” from LB(+)-isocitrate in
the protocol given above (see also Table I).
Results
The observation that extracts of aerobically grown pseudomonads
yielded from tricarboxylic acids a keto acid other than ketoglutarate and
pyruvate led to the chromatographic identification of this substance as
glyoxylate. The specific substrate and the stoichiometry of the reaction
could not be defined with the initial extract because of interfering enzymes
which equilibrated the tricarboxylic acids (aconitase) and those which
catalyzed further reactions of glyoxylate. Therefore, preliminary frac-
tionation of the extracts and a definition of the reaction were interde-
pendent.
Glyoqlic Acid; Identi$cation and Measurement-Glyoxylic acid was
identified as a product of the isocitritase reaction both by chromatography
and by the ultraviolet spectrum of its DiNPH. Glyoxylate formation
could be demonstrated by incubating 2 ml. of a sonic pseudomonad extract
(about 40 mg. of protein) at 30” under nitrogen, at pH 7.6, with any of the
tricarboxylic acids (citrate, cis-aconitate, or nn-isocitrate) (Fig. 1). The
dinitrophenylhydrazones of the keto acids formed in the enzyme reactions
and of pyruvic and glyoxylic acids, when chromatographed by the pro-
cedure of Cavallini et al. (15) as reported, each showed two well defined
spots. As observed in Fig. 1, the R, of the keto acid formed in the enzy-
matic cleavage of all three tricarboxylic acids corresponds to the DiNPH
of glyoxylate.
The ultraviolet absorption spectrum of the crystalline glyoxylic DiNPH
and the product formed enzymatically from L.(+)-isocitrate are shown in
Fig. 2, A, and the spectra of cr-ketoglutaric and pyruvic DiNPH in Fig.
2, B. As noted, the spectra of these three keto acid dinitrophenylhy-
drazones are readily distinguishable, and the spectrum of the reaction
product coincided with that of glyoxylic DiNPH. The extinction ratios
R. A. SMITH AND I. C. QUNSALUS 309
I I
_----- SO--L--V- ENT FRO-N---T-- ------ 1.00
FIQ. 1. Chromatographic identification of keto acids. Reaction contained in mi-
cromoles per 12 ml.; Tris buffer, pH 7.6, 400; substrate as shown, 25; extract 2 40
mg. of protein. Reaction for 45 minutes at 30” under NI, stopped with 0.5 ml. of 10
N HrSO, and the solution boiled for 5 minutes. The dinitrophenylhydrazones of the
keto acids formed were prepared by incubating the extract with an equal volume of
0.1 per cent DiNPH in 2 N HCl for 30 minutes at 37”. After extraction with ethyl
acetate, the dinitrophenylhydrazones were chromatographed by the procedure of
Cavallini ef al. (15).
Fro. 2. Ultraviolet absorption spectra of keto acid 2,4-dinitrophenylhydrazones.
A, glyoxylate and isocitritase reaction product; B, pyruvate and a-ketoglutarate.
2 #moles of keto acid in 1 ml. treated with 1 ml. of 0.1 per cent 2,4-dinitrophenylhy-
drazine in 2 N HCl for 30 minutes, diluted I:1 with 3.0 N NaOH and the spectrum
measured in a Beckman DU spectrophotometer. Glyoxylate DiNPH decomposes
rapidly, about 10 per cent per 10 minutes. Therefore extinction values are approxi.
mate.
310 ISOCITRITASE
490:540 rnp of the dinitrophenylhydrazones of these three keto acids differ
considerably; glyoxylic DiNPH gave a ratio of 1.9, and the pyruvic and
a-ketoglutaric dinitrophenylhydrazones gave ratios, respectively, of 1.1
and 1.0. Thus, in addition to determining the quantity of keto acid by
the direct method of Friedemann and Haugen (14), the light absorption
ratio indicated whether only glyoxylate or also other keto acids were present
in the mixture.
Suminic Acid; IdentiJication-An aliquot of the reaction mixture of
enzyme and L,(+)-isocitrate examined for glyoxylate in Fig. 2, A was
acidified and extracted for 8 hours in a continuous liquid-liquid extractor
with peroxide-free ether. 0.3 ml. of 0.02 M phosphate buffer, pH 7.0,
was added to the ether extract. The ether was evaporated and the residue
chromatographed, along with a 1 pmole sample of succinate treated simi-
larly, on Whatman No. 1 filter paper with n-butanol; 4 N formic acid
(60:40) as solvent. After removal of volatile acids by steaming, the
fixed acids revealed by 0.02 per cent chlorophenol-red spray were succinic
and isocitric.
Isocitritase
Purification-An extract of P. aeruginosa was fractionated for isocitritase
as shown in Table II, all the procedures being carried out at O-3” unless
otherwise specified. Ammonium sulfate was recrystallized from a slightly
ammoniacal 0.02 M Versene solution according to Beisenherz et al. (18).
The protein content was adjusted to 10 to 15 mg. per ml. for ammonium
sulfate fractionation, except after gel treatment, in which case the level had
been reduced to about 2 mg. per ml. by dilution with the gel. Protein
measurements in extracts with a 280:260 rnp light absorption ratio of
less than 1 were obtained by the TCA method of Stadtman et al. (19) and
in extracts with a 280:260 rnp ratio greater than 1 by calculation according
to Warburg and Christian (20).
A typical fractionation (Table II) was performed on 300 ml. of extract
prepared by sonic disintegration, and contained 17.5 mg. of protein and
22 isocitritase units per ml.; i.e., specific activity 1.25.
The first ammonium sulfate fraction containing isocitritase was prepared
by the slow addition with stirring of 46.5 gm. of solid ammonium sulfate
(0.22 saturation). The precipitate was collected by centrifugation and
discarded, and the supernatant fluid brought to 0.75 saturation by the
further addition of 113.5 gm. of solid ammonium sulfate under the same
conditions as before. The precipitate, ammonium sulfate I, when dis-
solved in 225 ml. of 0.02 M potassium phosphate buffer, pH 7.0 (containing
10B3 M cysteine and 10m4 M Versene), assayed 18 mg. of protein per ml.
and about 90 per cent of the initial isocitritase activity; specific activity =
1.6.
Ii. A. SMITH AND 1. C. GUNSALUS 311
Nucleic acid was removed from this solution, after the pH was adjusted
to 6.0 by dropwise addition of 1 N acetic acid with constant stirring, by
the slow addition of 0.1 volume (22 ml.) of protamine sulfate4 (20 mg. per
ml.; pH 5). The protamine nucleate precipitate was removed by centri-
fugation and left a supernatant solution with a light absorption ratio
280: 260 rnp of 0.9. Dialysis overnight against 0.01 M potassium phosphate,
pH 7.0, yielded an additional precipitate. After centrifugation, the
supernatant solution (PS) contained 16 mg. of protein per ml. and 85
per cent of the original isocitritase activity; specific activity = 1.8.
G&l treatment afforded further purification by adsorbing inactive protein.
The PS fraction was adjusted to pH 5.5 with 1 N acetic acid and 1 ml.
aliquots subjected to treatment with increasing amounts of calcium phos-
TABLE II
Fractionation of P. aeruginosa Extracts
Fraction Protein Isotacxit ri- aScpticvcitiyh c
-___
Extract*............................................... 5.22 6.55 1.25
Ammonium sulfate I, OX-O.75 saturation. . . 4.03 6.34 1.6
Protamine solution..................................... 3.01 5.41 1.8
Gel solution........................................... 0.78 4.50 5.8
Ammonium sulfate II, 0.43-0.56 saturation . 0.23 3.66 16.0
“ ‘1 III, OH-O.55 saturation. . 0.047 1.63 35.0
Protocol aa in Table I.
* Obtained by sonic disintegration of 60 gm. of cell paste; for the details, see
the text.
phate gel prepared according to Keilin and Hartree (21) (26 mg. of solids
per ml.). 0.7 volume of gel removed a maximal amount of protein without
appreciably adsorbing the isocitritase. Therefore, to the remaining PS
fraction (224 ml.), pH 5.5, were slowly added 158 ml. (0.7 volume) of
calcium phosphate gel, and the suspensionw as stirred gently for 20 minutes.
The gel was collected by centrifugation, and the supernatant solution (GS) ,
volume 363 ml., was found to contain 70 per cent of the original isocitritase
activity at a specific activity of 5.8.
A second ammonium sulfate fractionation achieved further purification
as follows: The GS fraction, 360 ml., 2.2 mg. of protein per ml., was treated
with 110 gm. (0.43 saturation) of ammonium sulfate, the precipitate was
discarded, and the supernatant solution was brought to 0.56 saturation
by further addition of 33 gm. of ammonium sulfate. The precipitate,
4 Supplied by Eli Lilly and Company.
312 ISOCITRITASE
ammonium sulfate II, when dissolved in 16 ml. of 0.02 M potassium phos-
phate buffer, contained 50 per cent of the original enzyme at a specific
activity of 16.
An alkaline ammonium sulfate precipitation of the ammonium sulfate
II brought the specific activity to 35. This was accomplished by adding
1 volume of saturated ammonium sulfate, adjusted to pH 7.5 and con-
taining lOA M Versene, to 1 volume of the ammonium sulfate II containing
10 mg. of protein per ml. The precipitate contained little activity and
so was discarded, and an additional 0.22 ml. (0.55 saturation) of the
saturated alkaline ammonium sulfate was added per ml. of original am-
monium sulfate II solution. The precipitate, ammonium sulfate III,
upon being dissolved in 0.02 M potassium phosphate buffer, pH 7.0, at 0.3
ml. per ml. of ammonium sulfate II fractionated, contained 25 per cent of
the original isocitritase at specific activity 35.6
The enzyme at this purity was reasonably stable in the frozen state,
-2O”, but upon storage for 9 months lost 75 per cent of its activity, which
was not restored by addition of cysteine or metals.
Activators-During the first attempts to fractionate isocitritase, the
enzymatic activity was usually lost after protamine treatment or upon
aging. The addition of cysteine or glutathione to the assay mixture in
the presence of MgCh, however, permitted nearly complete recovery of
activity. Dialysis of ammonium sulfate II (Table II) against 0.02 M
Versene, pH 7.4, for 20 hours at 5O, followed by 14 hours dialysis against
0.02 M KC1 at the same temperature, rendered isocitritase activity com-
pletely dependent upon metal and sulfhydryl compounds. The saturation
curves for Mg++, cysteine, and GSH (Fig. 3) show that GSH is less active
than cysteine, both in affinity and in the maximal activity reached. Fer-
rous and cobaltous ions restore about 40 per cent and manganous ions about
20 per cent of the activity given by magnesium ions at the concentration
of its maximal activity.
&enzyme A is not required for isocitritase action since treatment with
Dowex 1 chloride (22), under conditions which removed all the CoA from
the preparation, as measured by the transacetylase assay (19), did not
depress its activity.
Isoeitritase is active over a wide range of pH with a maximal activity
at pH 8.0 to 8.5 (Fig. 4). In most experiments, however, the reaction was
run at pH 7.6, where the reaction rate is nearly maximal, and glyoxylate,
which is alkali-labile, is less rapidly destroyed.
Stoichiometry-Fractionation of sonically prepared extracts to remove
6 Recently, in our laboratory Mr. H. H. Daron haa achieved about 1Wfold puri-
fication of isocitritase by this procedure.
313
R. A. SMITH AND I. C. GUNSALUS
the enzymes which catalyze side reactions permitted definition of the
isocitritase-catalyzed reaction as
Mg++, RSH
LB(+)-Isocitrate , ’ glyoxylate + succinate (1)
Although citrate, cis-aconitate, and isocitrate all serve as substrates with
fresh extracts, removal of aconitase either by aging or by fractionation
removed activity for all but isocitrate (Table III). Glyoxylate, as shown,
was stable in the presence of the purified enzyme preparations.
ISOCITRITASE - ACTIVATORS
2 ISOCITRITASE -
f!l I.0
p!
0 I 2 3 4 5 q”7 8 9 IO
/dvlOLES Mg*+or RSH / ML. PH
Fro. 3 FIG. 4
FIQ. 3. Isocitritase cofactor saturation. Reaction contained in micromoles per
1.5 ml.; Tris buffer, pH 7.6, 100; enzyme ammonium sulfate II (Table II) = 0.2 mg.
of protein; L.(+)-isocitrate, 5; MgCL, 3, or as indicated; RSH cysteine, 2.0, or as
indicated; reaction for 10 minutes at 30” under Nz, stopped by addition of 0.1 ml.
of 80 per cent TCA.
FIQ. 4. pa-dependence of isocitritase; protocol ZLY for Fig. 3; enzyme, ammonium
sulfate III = 0.11 mg. of protein. See Table II.
Substrate Afinity and Product Inhibition-The reaction rate curves
for enzyme saturation by L.( +) -isocitric acid and the influence of the prod-
ucts on the rate are shown in Fig. 5. A plot of the reciprocal of velocity
versus the reciprocal of L.(+)-isocitrate concentration was linear and
extrapolated to a Michaelis constant, K,, of 4.5 X 1O-4M . Both succinate
and glyoxylate inhibit isocitrate cleavage in a non-competitive manner,
as indicated from reciprocal plots of their concentration versu.st he reaction
rate. These constants, calculated by the Lineweaver-Burk (24) equation
for non-competitive inhibition, were 7 X lOmaM for succinate and 2 X lo-3
M for glyoxylate. Although the reaction is reversible, i.e. L.( +) -isocitrate
314 ISOCITRJTASE
is formed from glyoxylate plus succinate (see below), Michaelis constants
for glyoxylate and succinate as substrates were not measured.
Reversibility and h’quilibrium-The reversibility of this reaction was
suggested both by the initial experiment of Campbell and Smith (25) with
extracts of P. aeruginosa and by subsequent experiments of Olson (4) with
extracts of Penicillium chysogenum. Both groups reported an accumula-
tion of citrate upon incubation of the extracts with glyoxylate plus suc-
cinate. Presumably the citrate was formed by the combined action of
isocitritase and aconitase. To determine whether Reaction 1 was freely
TABLE III
Stoichiometry and Substrate Specijicitu
Fraction and substrate Added
pmolcs
Extract
Citrate ............................. 12.5 1.40
cis-Aconitate ....................... 12.5 4.44
nL-Isocitrate ....................... 20.0 8.28 7.27 8.35
Isocitritase (ammonium sulfate II)*
Citrate ............................. 12.5 Nil
cis-Aconitate ....................... 12.5 “
nL-Isocitrate ....................... 20.0 9.08 9.31 9.16
Reaction volume 3 ml., containing 5 units of isocitritase; reactants as in Table I
except for substrate. Succinate was determined with pig heart succinoxidase (23)
on an aliquot deproteinized by being boiled 5 minutes in 2 N HISO,. Glyoxylate was
determined on the TCA filtrate and isocitrate with isocitric dehydrogenaae aa indi-
cated under “Materials and methods.”
* See Table II.
reversible, isotope exchange and stoichiometric measurements were made
with the purified enzyme.
The catalytic incorporation of succinate-2,3-CL4 into isocitrate in the
presence of glyoxylate is shown in Table IV. With 0.005 M each of glyoxy-
late and succinate, slightly more than 1 pmole of isocitrate accumulated.
The Cl4 specific activity was equal, on a molar basis, to the succinate
added. Succinate and isocitrate were separated on a Dowex 1 column
(17) after forming glyoxylic DiNPH. The succinate recovery was quanti-
tative (8.8 + 1.2 pmoles), whereas 0.3 pmole of glyoxylate was not ac-
counted for (9.6 + 1.2 = 10.8 pmoles as compared to 11.1 rmoles added).
Since an excess of enzyme was used in this experiment and equilibration