THE JOURNAL OP Bxomolcn~ CHEMISTRY

Vol. 249, No. 22, Issue of November 26, pp. 714%71M), 1974
Printed in U.S.A.

Glutathione S-Transferase A
A NOVEL KINETIC MECHANISM IN WHICH THE MAJOR REACTION PATHWAY DEPENDS ON SUB-
STRATE CONCENTRATION

(Received for publication, May 1, 1974)

MICHAEL J. PABST,* WILLIAM H. HABIG, AND WJLLJAM B. JAKOBY

From the Section on Enzymes and Cellular Biochemistry, National Institute of Arthritis, Metabolism and Diges-
tive Diseases,National Institutes of Health, Bethesda, Maryland SO014

SUMMARY approximation we conducted separate experiments at both the

high and low regions of GSH concentration. At high levels of
Glutathione transferase A has been purified from rat liver.
GSH, approximately 0.15 to 5 mM, kinetic analysis indicated an
The enzyme catalyzes the conjugation of glutathione with
ordered sequential mechanism, with GSH adding first; a similar

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compounds bearing an electrophilic site, especially those in
ordered sequential mechanism was recently demonstrated for an
which the electrophilic site is on, or cr to, an aromatic ring.
enzyme conjugating menaphthyl sulfate with GSH (4). How-
The enzyme has a molecular weight of 45,000 and is composed
ever, in the GSH concentration range of 0.1 mM or lower, the
of two similar subunits.
data with glutathione transferase A suggested a ping-pong
Initial velocity, product inhibition, and binding studies
mechanism in which the electrophilic substrate added first.
indicate a biphasic kinetic mechanism in which the reaction
A rate equation has been developed for the system which is
pathway depends on the concentration of the substrates.
applicable to the entire range of GSH concentrations. The
At high concentrations of GSH, an ordered sequential path-
general form of the rate equation allowed a prediction which we
way predominates in which GSH binds first. At low con-
were able to verify experimentally (see “Appendix” (5)). Thus,
centrations of GSH, a ping-pong pathway predominates in
we were able to unify our observations with glutathione trans-
which the electrophilic substrate adds first. In accordance
ferase A into a concept of a dual pathway mechanism which
with a prediction of the general rate equation for the over-all
states that the flux is predominantly through the ordered path-
mechanism, the breakpoint in the biphasic double reciprocal
way at high GSH concentrations and predominantly through
plot for GSH saturation was found to shift to lower GSH con-
the substituted-enzyme pathway at low GSH concentrations.
centrations as the concentration of the electrophilic substrate
was lowered. A numerical rate equation was developed
MATERIALS AND METHODS
which describes initial velocities over the entire range of sub-
strate concentrations. An appendix presents a method for ChemicaZs-[7-14C]Benzyl chloride (250 &i, 13.92 mCi per
distinguishing among several formal kinetic mechanisms mmole: New Eneland Nuclear Corn.). a solution in 0.25 ml of
which yield nonlinear double reciprocal initial velocity plots as benzenk was dilited 1:lO into ethandi and stored at -15”. l-
Chloro-2,4-dinitro[U-Wlbenzene (50 &i per mmole, Amersham-
a result of multiple reaction pathways. Searle Corp.) was dissolved in 0.2 ml of ethanol prior to use.
S-(Z-Chloro-4-nitronhenyl)glutathione was prepared by a
modification of the published procedures (6, 7). Glutathione
(4.0 n1 was dissolved in 100 ml of 0.5 N H&O4 and heated to 50”.
&p&us oxide (0.95 g) was triturated with 3 ml of acetic acid, in
which it partially dissolved. The cuprous oxide suspension was
slowly added dropwise to the heated glutathione solution, re-
Glutathione S-transferase A is one of four similar enzymes
sulting in a silky white precipitate of the copper salt of gluta-
which we have purified to homogeneity in the course of an in- thione. 2-Chloro-4-nitroaniline (Aldrich Chemical Co.) (2.24 g)
vestigation into the glutathione-conjugating activity of rat liver was dissolved in 1 N H2S01 at 50” and filtered to remove insoluble
(l-3). Each of the glutathione transferases catalyzes the re- impurities. The solution was cooled to 0”, whereupon the
action of GSH with compounds bearing an electrophilic site (3). 2-chloro-4-nitroaniline precipitated, yielding a suspension of
flocculent crystals in a yellow solution. Sodium nitrite, 0.92 g,
The preparation and certain of the properties of transferase A dissolved in 2 ml of water, was added to the suspension. The
are described here. resultant colorless solution was slowly added to the copper gluta-
An initial kinetic evaluation of the enzyme revealed an un- thione suspension at 0”. Stirring was continued for 1 hour at 0”
expected complexity: the saturation with GSH in a double and additionally for 1 hour at room temperature. After filtration,
reciprocal plot was biphasic, yielding one apparent K, value for the filtrate was washed with a total of 750 ml of ether. Activated
charcoal was added to the aqueous phase, removed by filtration,
GSH in the high range of GSH concentrations and a different and washed with 1 liter of water. The product was eluted from
apparent K,,, value for GSH at low concentrations. As a first charcoal with 500 ml of methanol-aqueous ammonia (2O:l) and
concentrated to a yellow oil. After acidification with acetic
* Present address, School of Dentistry, University of Colorado acid. the oil was dissolved in 50 ml of water and warmed to 50”.
Medical Center, Denver, Cola. At &I”, ethanol was added until a slight cloudiness appeared.

7140
 7141

After cooling to -15’, the compound crystallized and was re- TABLE I
crystallized from water-ethanol.
The product was purified by chromatography on Whatman Summary of purijication

T
No. 3MM paper with I-butanol-acetic acid-water (12:3:5) to Assay by the standard system, with 1,2-dichloro-4-nitrobenzene
remove traces of both oxidized and reduced glutathione. Re- as substrate.
chromatography of a sample on an Eastman Chromagram-cellu-
lose sheet with the same solvent revealed a single spot (RF = 0.70) Purification Tota 1 Total Specific

T
which was ultraviolet-absorbing and reacted with ninhydrin.
step Volume protein activity activity
The final concentration of the product in aqueous solution was
determined by its absorbance (~(5 = 8.5 m&). -1 -1
ml Ez ttmoles min.l u min rng
The other compounds used were obtained commercially or were
recrystallized separately, as described (3). 1. Extract 1400 61,000 1100 0.018
Methods-For sucrose gradient centrifugation, the enzyme was
2. DEAE-Cellulose 1520 8,700 940 0.11
layered on top of a 5 to 20yo sucrose gradient in 0.1 M potassium
phosphate, pH 7.5, and centrifuged at 36,500 rpm for 26 hours in 3. Ammonium sulfate 190 7,100 860 0.12
an SW 39 rotor (8). Standard enzyme assays, sedimentation

I
4. m-Cellulose 10 330 250 0.75
equilibrium centrifugation, and sodium dodecyl sulfate-gel elec-
trophoresis were performed as described in the preceding paper 5. Hydroxylapacice 12 42 150 3.6

(3). 6. Hydroxylapatice 23 37 150 4.1

Preparation of Enzyme-The initial steps in preparation of
glutathione transferase A from rat liver have been described (3)
as part of a general method for separating the several related
enzymes. These steps include homogenizing the tissue and
applying the extract to DEAE and CM-cellulose columns. Frac- the same shape, a molecular weight of 45,000 was calculated for
tions 180 through 195 from the CM-cellulose column (see Fig. 2 of transferase A.
Ref. 3) were pooled and concentrated to 20 ml by ultrafiltration
Gel electrophoresis in sodium dodecyl sulfate produced a

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with a Diaflo PM-10 membrane.
The concentrated enzyme was dialyzed for 18 hours against single band corresponding to a molecular weight of 25,000 under
500 ml of 50 mM potassium phosphate, pH 6.7, containing 5 mM conditions in which six standard proteins allowed a linear plot of
GSH, 1 rnM EDTA, and 30% glycerol (Buffer B), with two changes mobility against log molecular weight.
of buffer. This material w&s applied to a column of hydroxyl-
apatite (4 X 20 cm) equilibrated with Buffer B. The column was
When subjected to gel filtration with Sephadex G-75, the
rinsed with 250 ml of Buffer B, followed by a linear phosphate elution position of transferase A was symmetrical and indis-
gradient consisting of 750 ml of Buffer B and 750 ml of the same tinguishable from that of an ovalbumin standard (45,000; D =
buffer, except that the concentration of potassium phosphate 0.75 cm8 g-l).
was 350 mM. Fractions of 12 ml were collected. A single major Stability-Under refrigeration, transferase A is stable for at
peak of activity, eluting in Fractions 70 through 79, was concen-
trated by ultrafiltration and dialyzed against Buffer B. least 6 months in 0.1 M potassium phosphate, pH 7.0, containing
The resultant protein solution was applied to another hydroxyl- 30% glycerol and 1 mM EDTA. The enzyme loses activity in
apatite column of the same size and under the same conditions as the presence of 2-mercaptoethanol, although other sulfhydryl
in the previous step and w&s eluted with the same gradient. The reagents, such as glutathione, thioglycerol, and dithiothreitol,
product of the second hydroxylapatite column was concentrated, act as stabilizing agents.
dialyzed against Buffer B, and stored at -80”.
Purified transferase A was crystallized from ammonium sulfate At pH 7.0, in the absence of glycerol and either EDTA or a
by the general method previously described (9), using successive sulfhydryl reducing agent, the enzyme gives rise to multiple
salt concentrations of 80, 75, and 67yo of saturation; a yield of forms which can be separated on hydroxylapatite or on isoelectric
80% was obtained at 670/, ammonium sulfate. Since crystalliza- focusing gels (11) in the range of pH 7 to 9 (Fig. 1). The differ-
tion did not increase the specific activity of the enzyme, it w&s
not used as a step in purification. ence in the isoelectric point (PI) between these forms is on the
order of 0.1 pH unit. The various forms are enzymatically
RESULTS active, sediment identically on a sucrose gradient, and are inter-
Puti$cation-A summary of the results of purification is convertible by dialysis in the presence or absence of the stabilizing
presented in Table I. A yield of 14y0 from the crude extract factors. The preparation used for further study was purified in
the presence of the stabilizing factors and appears as a single
was attained with the use of 1,2-dichloro-4-nitrobenzene as the
assay substrate; it should be recalled that transferase A repre- species upon chromatography with hydroxylapatite. Gel iso-
electric focusing in a pH range of 7 to 9 (11) resulted in one
sents only one of the enzyme species active with this substrate
major band which was accompanied by a faint secondary band
which are present in the extract (2, 3).
Homogeneity and Molecular Weight-The enzyme is homo- at a slightly lower isoelectric position. When the gel was sliced
and the isolated major band again was electrofocused, the second
geneous by the criteria of sedimentation equilibrium centrifu-
faint band reappeared. Both bands were enzymatically active.
gation, sucrose gradient centrifugation, and sodium dodecyl
sulfate-gel electrophoresis. Inactivation by Zodosobenzoate and by Zodoacetate-Since some of
the substrates of transferase A are classical sulfhydryl alkylating
Sedimentation equilibrium analysis in Buffer B at two concen-
agents, it was not surprising to find that iodoacetate alone was
trations of protein (50 and 100 pg per ml) resulted in linear plots
of r* versus the log of protein concentration. A molecular completely ineffective in inactivating the enzyme. However,
weight of 45,500 was calculated on the basis of a partial specific o-iodosobenzoate proved to be a good inactivator at a concentra-
volume of 0.737 cm’ g-1; the latter value was estimated (10) tion as low as 20 PM, although the time course of inactivation
from the amino acid composition (3) was unusual. The logarithm of the percentage of the remaining
Upon sucrose gradient centrifugation, a single symmetrical activity, when plotted against time, was not linear, indicating
peak of enzyme activity was observed at 3.5 S which corre- that the process was not a simple first order reaction, but rather
sponded exactly with that of horseradish peroxidase (40,000 consisted of an initial rapid inactivation down to 20 to 30% of the
daltons; partial specific volume (fi) = 0.699 cm8 g-l). On the initial activity, followed by a slower loss of the remaining ac-
assumption that the peroxidase and the GSH transferase have tivity (Fig. 2). Interestingly, when iodoacetate, ineffective
7142

r 100
90
12
90
70
60

OLC
20
--
/”25
m

FRACTION
30
NUMBER
35

FIG. 1. Chromatography on hydroxylapatite of transferase A

maintained in the absence of stabilizing factors. Enzyme which
had previously been dialyzed against 0.1 M potassium phosphate,
pH 7.5, and stored for several months at -30” was chromato- 0 IO 20 30 40
graphed on hydroxylapatite with Buffer B. The enzyme was
TME (MN)
eluted with a linear gradient of 200 ml of Buffer B plus 200 ml of
Buffer B containing 0.35 M potassium phosphate. Fractions (5 FIG. 2. o-Iodosobenzoate inactivation. At time zero, prior to

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ml) were collected, starting with the emergence of the gradient. assay with 1,2-dichloro-4-nitrobenzene, enzyme (0.04 FM in 0.1 M
The standard 1,2-dichloro-4-nitrobenzene assay was used (3). potassium phosphate, pH 7.5) was treated at 25’ with iodosoben-
The inset presents the results of gel isoelectric focusing in the zoate and substrates as indicated: A, enzyme alone; l , enzyme
pH range 7 to 9. a, Enzyme before addition of Buffer B, which with 0.02 mM o-iodosobenzoate; 0, enzyme with 1.0 mM o-iodoso-
contains the stabilizing factors; b, enzyme after addition of Buffer benzoate; 0, enzyme with 0.05 mM o-iodosobenzoate and 5 mM
B, prior to being applied to the hydroxylapatite; c, enzyme from GSH; A, enzyme with 0.05 mM o-iodosobenzoate and 1 mM 1,2-
Fraction 27; d, enzyme from Fraction 30. dichloro&nitrobenzene; n , enzyme with 0.05 mM o-iodosoben-
zoate and 2 mM iodoacetate.
alone, was included with o-iodosobenzoate, inactivation went
rapidly to completion. 1
Saturating levels of glutathione protected the enzyme to a
substantial degree, probably by reacting with the o-iodoben-
zoate. However, 1,2-dichloro-4-nitrobenzene also protected the
enzyme; whether it did so by reacting with the o-iodobenzoate
or by binding to the enzyme was not determined (Fig. 2).
Sulfhydryl Group Titration-The enzyme was titrated with
5,5’-dithiobis(2-nitrobenzoate) (12), yielding 3.5 sulfhydryl
groups per enzyme. Titration of sulfhydryl groups was closely
paralleled by loss of enzymatic activity. Unfortunately, 1,2-
dichloro-4nitrobenzene and the other aromatic substrates inter-
PH
fere with the reaction, so that it was not possible to obtain
meaningful data with the sulfhydryl reagent in the presence of FIG. 3. pH dependence of conjugation in the absence of enzyme
(closed symbols) and in the presence of 35 nM enzyme, after cor-
substrates. rection for the nonenzymatic reaction (open symbols): 0, 0,
Effect of pH-Fig. 3 shows the pH dependence of the non- p-nitrobenzyl chloride; 0, H, 1,2-dichloro-4-nitrobenzene; A,
enzymatic and the corrected enzymatic rates for three substrates. A, 4-nitropyridine N-oxide. Buffers were 0.1 M in the following
Whereas the pH at which the nonenzymatic rate becomes pH ranges: potassium phosphate, pH 6 to 7.8; Tris-chloride, pII
significant is quite different for each of the compounds, the 7.8 to 9.0; potassium glycine, pH 9 to 11.
enzyme allows reaction of all three with GSH in the physiological
range of pH. absorbance, indicating that reversibility is less than 1%. Simi-
Stoichiometry and Reversibility-The conversion of 1,2-di- larly, in the absence of GSH, [7-i4C]benzyl chloride (0.1 mM;
chloro-4-nitrobenzene to S-(2.chloro-4-nitrophenyl)glutathione 13.92 mCi per mmole) was not converted to S-benzylglutathione
is stoichiometric. 1,2-Dichloro-4-nitrobenzene (25 and 50 in the presence of S-(2-chloro-4-nitrophenyl)glutathione (0.1 mM)
nmoles), in the standard assay system, was completely converted and 3 pg of enzyme in 1.0 ml of 0.1 M potassium phosphate, pH
to S-(2-chloro-4-nitrophenyl)glutathione, as judged by the 7.5; addition of 0.1 mM GSH to this system produced the expected
change in absorbance at 345 nm. The extinction coefficient of S-benzylglutathione. Incubation mixtures were separated by
the product at 345 nm is 8.5 mM-1, which is the same as the AE chromatography on Whatman No. 1 paper with l-butanol-
for the reaction, since the substrate does not absorb significantly acetic acid-H20 (12:3:5) and assayed for radioactivity with a
at 345 nm. Vanguard strip counter.
The reaction is experimentally irreversible. Transferase A Substrate Specificity-In the presence of sat’urating GSH (5
(3 pg) was incubated with 10 PM S-(2-chloro-4-nitrophenyl)- mM), 1,2-dichloro-4-nitrobenzene, 4-nitropyridine-N-oxide, and
glutathione in the absence of GSH in 1.0 ml of 0.1 M Tris-chloride, p-nitrobenzyl chloride showed apparently normal hyperbolic
pH 7.5. No change in Asd5 was detectable with a Cary 15 saturation of transferase ,4, with K, values in the region of 1
spectrophotometer adjusted for a full scale deflection of 0.1 mM (Table II). I3ecause of their low solubility, it was not
 7143

TABLE II
Specijic activities and kinetic parameters for various substrates
I
Specific 6
16
”
Substrate activit; Knl max 1
12 6
-1 >
moles min- m /
6
1
4
!!a2 ,le enzyme

1. 1,2-Dichloro-4-nitrobenzene 1.10 390

I ,
2. p-Nitrobenzyl chloride 1.35 1200 0 .2 4 .6
IItBsPl l/AM’l
3. 4-Nitropyridine-N-oxide 1.05 160
FIG. 4 (left). Substrate inhibition by BSP in the spectrophoto-
4. 1,2-Epoxy-3-(p-nitrophenoxy)-props metric assay(3), usinga 5-cmlight path and 35 nM enzyme.
5. 1,2-Naphthylene oxide
FIG. 5 (righl). BiphasicapparentK, for GSH in the otherwise
standard assaysystemwith 1,2-dichloro-4-nitrobenzene.
6. Methyl iodide

7. 1-Memphthyl sulfate I/[GSHl(mM-‘1 I/Cl,2-DICHLORO-4-NE~E~l(mM-~~

0 2 4 6 80 2 4 6 0 IO
8. trans-4-Phenyl-3-butene-Z-one 3.21 I J/I- 0 n

9. p-Nitrophenethyl bromide

10. Bramosulfophchalein 3.002 72

11. 1-Chloro-2,4-dinirrobenzene 60.0 0.06 3000

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= Under standard assay conditions (3).
it Apparent Km since CSH is not saturating under standard assay conditions.

possibleto achieve concentrations of these compoundsgreater

than 1 mM. At a lower concentration of GSH (0.25 mM), chosen
to minimize the nonenzymatic reaction, Irons-4-phenyl-3-buten-
a-oneshowednormal saturation at concentrationsas high as 0.6
rnM. 1-Chloro-2,4-dinitrobenzenealsodisplayednormal satura-
tion, with a K, of 0.06 mM with 1 mM GSH. The enzyme is
also somewhatactive with naphthaleneoxide, p-nitrophenethyl
bromide, and 1,2-epoxy-3-(p-nitrophenoxy)propane but does
not catalyze conjugation with methyl iodide or menaphthyl
sulfate (Table II). Other glutathione transferaseshave higher
specific activities with these last five substrates(2, 3). Com- 0’ I I
parisonof the relative ratesof reaction from this limited sampling 0 20 40 60 800 I 2 3 4 5
I/[GStlI (mM-I) I/~l,2-DICHLORO-4-N~~~l~mM-~~
of substratesindicatesthat this enzyme favors substrateswhose
leaving group is on, or (Yto, the aromatic ring. FIG.6. Initial velocity and rate equations. A and B, initial
velocity data in the high range of GSH concentrations. In A,
Becauseof its clinical interest asan indicator of liver function, the concentrations of 1,2-dichloro-4-nitrobenzene were: a, 0.1
bromosulfophthaleinwas tested as a substrate with a spectro- mM; b, 0.2 mM; c, 0.4 mM; d, 0.6 mix; e, 1.0 mM. In B, the concen-
photometric assay(3). The compounddisplaysstrong substrate trations of GSH were: f, 0.15 mM; g, 0.2 mM; h, 0.3 mM; i, 0.5 mM;
inhibition (Fig. 4), with a K, of 2 PM and maximal activity in j, 1.0 mM. C and D, initial velocity data in the low range of GSH
concentrations. In C, the concentrations of 1,2-dichloro-4-nitro-
the region of 5 FM. In D,
benzene were: k, 0.2 mM; 1, 0.3 mM; m, 0.4 nM; n, 0.6 mM.
GSH Saturation-A double reciprocal plot of GSH saturation the concentrations of GSH were: o, 0.015 mM; p, 0.025 mM; Q, 0.05
was biphasic, with a discontinuity near 0.1 mM GSH (Fig. 5). mM; r, 0.10mM. Transferase A (0.16 rg) was used in each assay.
At concentrations higher than 0.15 mM, GSH shows normal Assays were performed in 1.0 ml of 0.1 M potassium phosphate,
pH
hyperbolic saturation, yielding an apparent K, of 0.2 mM. In tion7.5. The lines are calculated from the appropriate rate equa-

the region below 0.1 mM, saturation is also linear in the double ’
reciprocalplot, yielding an apparent K, of 0.01 mM. The same V,,, 0.267 2.10 0.107
biphasic curve was obtained when this saturation experiment V = ’ + [GSHI + -[DCNBI + [GSHICDCNBI
(1)

wasrepeatedin the presenceof 1 mu dithiothreitol.

Equation 1 indicatesK, valuesof 0.27 mMfor GSH and 2.1 mM
Rate Equations for High GSH Concentrations-Fig. 6, A and
for 1,2-dichloro-4-nitrobenzene(DCNB).
B, presentsdouble reciprocal plots of the effect of substrate Inhibition by Products at High GSH-Under normal assay
concentrations on the initial velocity. The range of GSH conditions, chloride ion was not inhibitory when tested at con-
concentrationsused was limited to that greater than 0.15 mM, centrations as high as 100 ~-IMwith GSH at saturation or with
i.e. thoseconcentrationsabove the break in the saturation curve. GSH near its K, level (0.05 mM). The other product, S-(2-
The range of 1,Zdichloro-Pnitrobenzene was limited by low chloro-4-nitrophenyl)glutathione, is a potent inhibitor. Fig. 7
solubility to concentrations below its K,,,. Despite these re- indicates that this compound is competitive with GSH, with a
strictions, the data produce an intersecting pattern of linesfrom Ki of 5 PM. The inhibition is noncompetitive with 1,2-di-
which the rate equation (Equation 1) wasdeveloped (13). chloro-4nitrobenzene (Fig. 7).
7144
[S-(2-CWORO-4-NIlROP+ENYL)-GLUTATWNEl [BENZYL ~HLORIDEI (mM) [SENZYL cti~cfu~~l
hM)
0 .a .02 0 .a .02

, I I I
0 02 04 .u? .04
h%
IS-(2-CKORO-4-NITR~NYL)-~~ATHK)NEI 0 0.5 1.0 0 0.5 1.0

FIQ. 7. Inhibition by S-(2-chloro-4-nitrophenyl)glutathione at [BENZYL CHLORIDE] htd [SENZYL c~~oRtoE1

high concentrations of GSH. A and B, secondary plots of non- FIG. 8. Inhibition by benzyl chloride at high concentrations of
competitive inhibition with 1,2-dichloro-4-nitrobenzene as the GSH. A and B, secondary plots of noncompetitive inhibition

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variable substrate (0.6 to 0.1 mM) and GSH concentration con- with GSH ae the variable substrate (1.0 to 0.10 mM) and 1,2-
stant at 0.2 mM. C and D, secondary plots of competitive inhi- dichloroAnitrobenzene constant at 1 mM. C and D, secondary
bition with GSH as the variable substrate (1.0 to 0.15 mM) and plots of noncompetitive inhibition with 1,2-dichloro&nitroben-
1,2-dichloro-4-nitrobenzene concentration constant at 0.5 mM. zene as the variable substrate (1.0 to 0.15 mM) and GSH concen-
tration constant at 5 mM.
Inhibition by Benzyl Chloride at High GSH-Benzyl chloride
is a substrate for the enzyme; the reaction may be followed
titrimetrically at pH 6.5. The production of S-benzylgluta-
thione was confirmed chromatographically. With 1 mM benzyl
chloride, the rate of production of S-benzylglutathione was 1.2
pmoles min-i mg-i of protein, a rate in the range found with
many other substrates in spectrophotometric assays (Table II).
Because benzyl chloride did not interfere with the spectrophoto-
metric assays and was available in radioactively labeled form
for binding experiments, its behavior as an inhibitor of the
standard 1,2-dichloro-4nitrobenzene reaction was studied.
Surprisingly, benzyl chloride was found to be noncompetitive
with both 1,2-dichloro-4-nitrobenzene and GSH (Fig. 8).
Inhibition by trans-&Phenyl-S-buten-g-one at High GSH-
Because of the unexpected results of inhibition with benzyl
chloride, another substrate which did not interfere with the 0 02 0.4 0 02 04

1,2-dichloro-4-nitrobenzene assay was tested as an inhibitor. [TRANS-4-PHENYL-3-BUTEN-2-ONE1 (mM)

trans-4-Phenyl-3-buten-2-one was competitive with 1,2-di- FIG. 9. Inhibition by trans.4-phenyl-3-buten-2-one at high
chloro-4-nitrobenzene and uncompetitive with GSH (Fig. 9). concentrations of GSH. A and B, secondary plots of competitive
inhibition with 1,2-dichloro-4-nitrobenzene as the variable sub-
Rate Equation for Low GSH Concentrations-Reliable initial
strate (1.0 to 0.15 mM) and GSH concentration constant at 5 mM.
velocity data are difficult to obtain in the region of low GSH C and D, secondary plots of uncompetitive inhibition with GSH
concentrations (between 0.015 mM and 0.1 mM) because of the as the variable substrate (1.0 mM to 0.10 mM) and 1,2-dichloro-4-
low velocities involved. Nevertheless, a rate equation (Equation nitrobenzene concentration constant at 1 mM.
2) could be calculated from the data shown in Fig. 6, C and D.
V difficult to distinguish competitive from mixed noncompetitive
max = 1 + 0.119 1.41 0.00925
Y iGssl + m + tGSHj[DCNBJ (2) inhibition when 1,2-dichloro-4-nitrobenzene was the variable
substrate. However, after repeated inhibition studies, we
The K, values are similar to those obtained in the high GSH found that at low GSH concentrations (0.03 mM), S-(2-chloro-4-
region, i.e. a K, of 0.12 mM for GSH and of 1.4 mM for 3,4- nitrophenyl)glutathione is competitive with 1,2-dichloro-4-nitro-
dichloronitrobenzene. However, the numerator of the last benzene; the data are presented in Fig. 10 for evaluation. The
term in the equation for the low GSH region is 1 order of magni- Ki for the product was 4 pM, which is the same as that deter-
tude smaller than that for the high GSH region. Because of this mined at high GSH concentrations. With GSH at low concen-
small numerator, the lines described by the equation in Fig. 6, trations (i.e. less than 0.1 mM) as the variable substrate, product
C and D, appear to be parallel, or nearly so. inhibition was of the mixed noncompetitive type (Fig. 11).
Inhibition by Product at Low GSH-Because of the low solu- Binding of I?‘-%‘]Benzyl Chloride-Enzyme was incubated in
bility of 1,2-dichloro-4-nitrobenzene, it was not possible to the presence of 0.65 mM radioactive benzyl chloride for 15 min
achieve concentrations of this compound which were greater at room temperature, and the mixture was passed through a
than its K,. In product inhibition studies, therefore, it was column of Sephadex G-75 to remove free benzyl chloride (Fig.
 7145

12A). The amount of radioactivity which remained bound to mixture was then applied to a column of Sephadex G-75, with
the enzyme corresponded to 0.9 mole of benzyl chloride bound the results shown in Fig. 13A : 0.40 mole of 1-chloro-2,4-dinitro-
per mole of enzyme. The labeled enzyme was divided into two benzene was bound per mole of enzyme. The fraction contain-
portions, one of which was reapplied to the Sephadex column. ing the peak enzyme activity was divided into three parts.
Approximately one-half of the benzyl chloride remained bound, One part was incubated for 5 min at 25’ with 1 mM dithio-
i.e. 0.45 mole of benzyl chloride per mole of enzyme (Fig. 12B). threitol and then was reapplied to the Sephadex column. The
The second portion of labeled enzyme was treated with 5 mM second part was incubated with 1 mM GSH and the third was
GSH before being reapplied to the Sephadex column. The incubated without any addition. The results are shown in Fig.
radioactive benzyl chloride was completely separated from the 13. The untreated enzyme retained 0.32 mole of l-chloro-2,4-
enzyme by GSH (Fig. 12C). dinitrobenzene per mole of enzyme. The sample treated with
Binding of 1 -Chloro-6,4-dinitro[ U-Vjbenzene-Enzyme (33 GSH, however, was reduced to 0.14 mole of substrate per mole
FM) was incubated with 0.57 mM radioactive 1-chloro-2,4-dini- of enzyme. Dithiothreitol was less effective (0.22 mole of sub-
trobenzene for 15 min at 25” in 1.0 ml of 0.1 M potassium phos- strate per mole of enzyme) than GSH in releasing bound sub-
phate (pH 7.5) containing 0.1 mM EDTA. The incubation strate.
Experimental Verification of Prediction of Over-all Rate Equa-
tion-Because of the difficulties associated with kinetic analysis
A in the presence of low concentrations of GSH and because we
12 wished to determine whether our separate observations at high
and low ranges of GSH concentration were consistent with a
single over-all mechanism, we consulted Dr. John Westley, who
IO developed the general over-all rate equation described as Scheme

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1 under “Appendix” (5). The general rate equation predicts
that the position of the apparent “breakpoint” in the double
reciprocal plot of GSH saturation is a function of the concen-
a
tration of the second substrate. Specifically, the concentration
of GSH at which the breakpoint occurs will be lower as t,he
concentration of 1,2-dichloro-4-nitrobenzene is decreased.
5 6 This prediction may be rationalized in the following way: as
1,2-dichloro-4-nitrobenzene is decreased, the pathway which
requires that this compound bind first becomes relatively less
4 significant when compared with the pathway in which GSH
binds first. The results of an experiment to test this prediction
are shown in Fig. 14. At 1 mM 1,2-dichloro-4-nitrobenzene,
the breakpoint (indicated by the intersection of the broken lint~)
2 appears to be in the region of 0.25 mM GSH, whereas at 0.6, 0.4,
0.2, and 0.1 mM 1,2-dichloro-4-nitrobenzene the breakpoints are
near 0.20, 0.13, 0.08, and 0.06 mM GSH, respectively, in accord
with the prediction.
-2 0 2 4 6 8 IO Correlation of Experimental Data with Numerical Rate Equa-
tion-The curves drawn in Fig. 14 were generated from the initial
I/[&2-DICHLORO-4-NITROBENZENEI (mM-‘I
velocity equation shown in Scheme 1. This equation is an
FIG. 10. Inhibition by S-(2-chloro-4-nitrophenyl)glutathione expansion of the general rate equation given in Scheme 1 of
at low GSH concentration. Competitive inhibition with 1,2-
dichloro-4-nitrobeneene as variable substrate and GSH concen- “Appendix,” and was derived by the method of King and
tration constant at 0.03 mM. Altman (14) from Scheme 2.

k_lkjkt+k,l‘k -2’k4’C + kZk3k4k2’k3’k4’AG + k-lk3k4k l’k3’k4’C + k2k3k4k I’k3’k4’C + k-lk3k4k2’k3’k4’AG

‘k 4 ‘G + klk3k4k-l’k-2’k4’AG + klk3k4ksl’ k 3 ‘k 4 ‘AC + klk3k4k2’k3’k4’A2G + klk2k 3k2’k3’k4’A2

+ k*kjk4k- 1 ‘k-q

+ klk*kqk-1 ‘k -2’k4’A + k k k k ‘k 3 ‘k 4 ‘A + klk2k4k2’k3’k4’A’ + klk2k3k l’ke2’k4’A

1 2 4 -l’k3’k4’A + klk2k-3k-l

+ klk2k3k2’k3’k4’A2G + k k k k ‘k ‘k ‘AG + klk2k3ksl’ks2’k4’AG + k-lk3k4kl’k-2’ k 4 ‘G* + kslk3k4kl’k3’k4’G’

123-1 3 4

+ k2k3k4kl’k3’k4’G2 + k2k3k4kl’ks2 ‘k 4 ‘G’ + k-lk3k4kl’k2’k4’AG” + k2k3k4kl’k2’k4’AG2 + k2k3k4kl’k2’k3’AG2

+ kelk3k4kl’k2’k3’AG2
E
2,
Y

k4[klk2k3k2’k3’k4’A*G + klk2k3ksl’k3’k4’AG + klk2k3ksl’kw2 ‘k/AC]

4 + k4’[k2k3k4kl’k2’k3’AG2 + k-lk3k4kl’k2’k3’Ati]

SCHEME 1
7146

.8

.6 .6

4 .4

.2 .2

4 18

.8 -
C
- 4.0

.6 3.0

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4 2.0
9,

.2 1.0

0 0

.8 4.0

.6 3.0

012345 ‘t-f-8- 10 I2 I4 I6 I8 20

[PRODUCT] (/.A) FRACTION NUMBER FRACTION NUMBER

FIG. 11 (left). Secondary plots of noncompetitive inhibition C, the remaining half of the enzyme was treated with 5 mM GSH
by S-(2chloro-4-nitrophenyl)glutathione with GSH as the vari- before being reapplied to the column.
able substrate (0.1 to 0.015 mM) and 1,2-dichloro-4-nitrobenzene FIG. 13 (right). Binding of 1-chloro-2,4-dinitro[U-“C]benzene.
concentration constant at 0.5 mM. A, elution profile on Sephadex G-75 of 33 nmoles of enzyme incu-
FIN. 12 (center). Binding of beneyl chloride. A, elution profile bated for 15 min at 25” with 0.57 mM 1-chloro-2,4-dinitrobenzene.
on Sephadex G-75 of 14 nmoles of enzyme, incubated for 15 min The peak fraction from A was split into three parts, and each
at 25” with 0.65 mM [7-r4C]beneyl chloride in 0.4 ml of 0.1 M potas- part was then incubated with the indicated addition and reapplied
sium phosphate (pH 7.5) containing 30% glycerol. (Enzyme to the Sephadex column. B, incubation with 1 mM dithiothreitol;
had been previously freed from GSH by gel filtration.) B, one- C, with 1 mM GSH; D, with water.
half of the enzyme from the peak in A was reapplied to the column.

used in the initial velocity equation to produce the curves

shown in Fig. 14. Although these constants are compatible
with the experimental data, they are only estimates and have
not been uniquely determined. Nevertheless, the mathe-
matically constructed curves in Fig. 14 correlate well with the
experimental data.
This numerical rate equation also indicates that no curvature
will be apparent in a double reciprocal plot of 1,2-dichloro-4-ni-
trobenzene saturation, provided that the concentration of
1,2-dichloro-4-nitrobenzene does not exceed its K, (2 mhf).
Since the limit of solubility of this compound is 1 mM, the appar-
In the diagram and rate equation, G represents GSH, A repre- ently normal hyperbolic saturation observed with this com-
sents 1,2-dichloro-4-nitrobenzene, C represents S-(2-chloro-4-ni-
pound is also compatible with the proposed mechanism.
trophenyl)glutathione, and a represents Cl-. Rate constants
were estimated from the experimental data of Fig. 14: kl = 2500 DISCUSSION
min-r kz = 150 min-r, k3 = 7.5 X lo6 min-r M-*, k4 = 150
M-I,
min-1, km1= 15 min-1, km3= 3 x lo4 min+, k’, = 1 x 10’ min-r Glutathione transferase A, which has been prepared in a
~-1, k’, = 1.6 x 104 min-r M-I, k’a = 20 mm-l, k’r = 30 min-l, homogeneous form, is characterized by a high turnover number
k’-l = 2 min-r, and k’-z = 58 nun-r. These constants were with compounds the electrophilic site of which is on, or (Y to, an
 7147

that 1,2-dichloro-4-nitrobenzene protected the enzyme against

inactivation by o-iodosobenzoate, offering the possibility that
the aromatic substrate would bind to the enzyme in the absence
of GSH. Subsequently it was shown that addition of 1 mM
benzyl chloride, in the absence of GSH, produced a 5% decrease
in the intrinsic fluorescence of the protein (excitation, 230 nm;
emission, 355 nm). The binding and release by GSH of radio-
active benzyl chloride and 1-chloro-2,4-dinitrobenzene support
the idea of a substituted enzyme at low GSH. However, it
must be admitted that the binding detected with radioactive
electrophilic substrates may not necessarily be mechanistically
significant, especially in view of the failure of GSH to release all
of the bound l-chloro-2,4dinitrobenzene. Since 1-chloro-2,4-
dinitrobenzene does inactivate the enzyme with time, it is
suggested that this reagent can alkylate the enzyme irreversibly.
Thus, the 1-chloro-2,4-dinitrobenzene which cannot be removed
by GSH may be due to nonspecific alkylation, whereas that
which is removed may be bound as substrate.
Two other kinetic observations deserve comment. The first
is that benzyl chloride is noncompetitive with both GSH and
1,2-dichloro-4nitrobenzene. This behavior may be due to

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benzyl chloride’s forming a complex with free enzyme as well as
I I 1 1 I
0' with enzyme-GSH complex. The second observation is that
IO 20 30 40 50
BSPl shows strong substrate inhibition. BSP has a compara-
I/[GSH](mW')
tively low K,,,, so that it is possible to approach saturation with
FIG. 14. Initial velocity plot in the region of transition be- this compound. Saturating concentrations of BSP would tend
tween the ordered sequential and ping-pong pathways, demon-
strating the movement of the breakpoint in GSH saturation to bind to free enzyme as well as to enzyme-GSH complex, even
toward lower concentrations of GSH as the concentration of in the presence of relatively high GSH concentrations. If the
1,2-dichloro-4-nitrobeneene is decreased from 1 mM (O ), to 0.7 velocity through the ping-pong pathway were slower than that
mM (O), 0.4 mM (O), 0.2 mM (A), and 0.1 mM (m). The curves through the ordered sequential pathway, substrate inhibition
are drawn from the numerical rate equation described in the text. would result. Substrate inhibition by BSP, viewed in this way,
The dashed lines indicate v.isual estimates of the position of the
breakpoint. confirms the ordered rather than random nature of the sequential
pathway.
aromatic ring. The enzyme is a dimer of 45,000 molecular Because of the complexity of the proposed mechanism, we
weight which requires approximately four sulfhydryl groups for tested whether a rate equation developed for the formal mecha-
activity. nism would be compatible with our initial velocity observations
This enzyme displays a complex kinetic mechanism. Under at both high and low GSH concentrations. The general rate
conditions extant in normal rat liver, i.e., in the presence of equation also yielded a prediction on the behavior of the GSH
between 3 and 10 mM GSH (15), transferase A activity reflects breakpoint. As demonstrated in Fig. 14, both the initial
the kinetics observed for the region of a high K,,, for GSH (Fig. velocities and movement of the breakpoint are consistent with
6, A and B). In this range, an ordered sequential pathway the formal mechanism.
predominates in which glutathione is the first substrate to react Enzymes generally have a single reaction pathway which is
with the enzyme. This is shown by the intersecting initial the most efficient pathway for a specific catalytic function.
velocity plots in the high GSH region, together with the stoichio- (An exception of which we are aware is human hypoxanthine
metric nature of the reaction, which rules out side reactions phosphoribosyltransferase, which also displays a dual, ordered,
(16). The order of addition is derived from the finding that sequential ping-pong mechanism (17). In this case, the pri-
the product, S-(2chloro-4-nitrophenyl)glutathione, is competi- mary ping-pong mechanism is supplanted by a ternary complex
tive with GSH and noncompetitive with the other substrate, mechanism when Mg*f is limiting.) The complex dual pathway
1,2-dichloro-4-nitrobenzene. Also in accord with the proposed mechanism of glutathione transferase A may be a consequence of
order of binding is the finding that trans-4-phenyl-3-buten-2-one, the need to maintain maximal flexibility in substrate specificity
a competitive inhibitor with respect to 1,2-dichloro-4-nitro- for this enzyme, the presumed function of which is to intercept
benzene, is uncompetitive with GSH. harmful compounds of unknown structure. A highly refined
Under conditions of low GSH concentrations, the initial and efficient single pathway mechanism may not be compatible
velocity pattern is that of a series of nearly parallel lines (Fig. with the necessary substrate flexibility.
6, C and D), suggesting an emerging ping-pong mechanism. The kinetic mechanism of glutathione transferase A is an
At low GSH concentrations, S-(2-chloro-4-nitrophenyl)gluta- example of a system in which nonhyperbolic kinetics, displaying
thione is competitive v+ith 1,2-dichloro-4-nitrobenzene and apparent “substrate activation” or “negative cooperativity,”
noncompetitive with GSH, a finding in accord with the idea mav be explained purely as a result of multiple reaction path-
that the electrophilic substrate is the first to bind at low GSH ways, without the need to invoke allosterism, conformational
concentrations. Since reliable kinetic data are difficult to obtain changes, or association-dissociation phenomena. This and other
in this region of low activity, i.e. at 0.1 mM GSH or lower, con- examples of multiple pathway mechanisms are discussed more
firmation of the ping-pong hypothesis was sought in binding fully under “Appendix” (5).
experiments in the absence of GSH. It had been observed r The abbreviation used is: BSP, bromosulfophthalein.
7148

REFERENCES 8. MARTIN, R. G., AND AMES, B. N. (1961) J. Biol. Chem. 236.

1372-1379
1. FJELLSTEDT T. A., ALLEN, R. H., DUNCAN, B. K., AND JA- 9. JAKOBY, W. B. (1971) Methods Enzymol. 22, 248-252
KOBY, W. B. (1973) J. Biol. Chem. 248, 3702-3707 10. COHN, E. J., AND EDSALL, J. T. (1965) Proteins, Amino Acids
2. PABST, M: J., HABIG, W. H. AND JAKOBY, W. B. (1973) Bio- and Peptides, p. 375, Hafner Publishing Co., Inc., New York
them. Biophys. Res. Commun. 62, 1123-1128 11. WRIGLEY, C. W. (1971) Methods Enzymol. 22, 559-564
3. HABIG, W. H., PABST, M. J. AND JAKOBY, W. B. (1974) J. 12. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82,7&77
Biol. Chem. 249, 7130-7139 13. CLELAND, W. W. (1963) Biochim. Biophys. Acta 67, 104-137
4. GILLHAM, B. (1973) Biochem. J. 136, 797-804 14. KING, E. L., AND ALTMAN, C. (1965) J. Phys. Chem. 60, 1375
6. WESTLEY, J., AND TAYLOR, H. (1974) J. Biol. Chem. 249,7148- 1378
15. TATEISHI, N., HIGASHI, T., SHINYA, J., NARUSE, A., AND
7150 SAKAMOTO, Y. (1974) J. Biochem. (Tokyo) 76, 93-103
6. BOOTH, J., BOYLAND, E., AND SIMS, P. (1960) Biochem. J. 74, 16. CHUNG, S. I., AND FOLK, J. E. (1972) J. Biol. Chem. 247, 2798-
117-122 2807
7. BOOTH, J., BOYLAND, E., AND SIMS, P. (1961) Biochem. J. 79, 17. KRENITSKY, T. A., AND PAPAIOANNOU, R. (1969) J. Biol.
616-524 Chem. 244, 1271-1277

APPENDIX

DISTINCTION AMONG FORMAL MECHANISMS THAT YIELD DOUBLE RECIPROCAL PLOTS CONCAVE

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FROM BELOW*

(Received for publication, May 1, 1974)

JOHN WE~TLEY AND HUGH TAYLOR

From the Department of Biochemistry, The University of Chicago, Chicago, Illinois 60637

Steady state data for several enzymes with double displace- are capable of generating either convex or concave curvature in
ment capability, i.e. the capacity to form a substituted enzyme double reciprocal plots for the acceptor substrate, i.e. the second
as an intermediate in the catalytic cycle, have yielded double substrate entering the double displacement cycle, depending on
reciprocal plots for one or more substrates that curve downward the values of the various rate constants and the accessible con-
as they approach the ordinate.1 Inspection of the initial velocity centration ranges of the substrates. In experiments in which
patterns in such cases can be used to distinguish among the curvature concave from below is obtained, the “breakpoint,”
several formal mechanisms that have been suggested to explain which marks the change from flux mainly through the double
this behavior. displacement to flux mainly through the single displacement, is
seen to vary with concentration of the fixed substrate, appearing
COMBINED SINGLE-DOUBLE DISPLACEMENT FORMS to drift off to the left as that concentration is increased (Fig. 1).
The same order and opposite order forms are distinguishable
A formal mechanism which combines single and double dis-
from each other in several ways. The criterion that is simplest
placement cycles has been proposed by Pabst et al. (1) for
in principle is based on the fact that mechanisms in which the
glutathione transferase A. This mechanism is given in its
substrates enter the single and double displacement cycles in
simplest form in Scheme 1.2 The similar formal mechanisms
opposite order (Scheme 1) can generate nonlinear double re-
in which the substrates enter the single and double displacement
ciprocal plots for the donor substrate as well as for the acceptor.
cycles in the same order rather than in the opposite order (Scheme
In contrast, the mechanisms in which the substrates enter the
2) have been discussed briefly as theoretical possibilities in an
single and double displacement cycles in the same order (Scheme
earlier publication (2). All of the combined single-double forms
2) yield only linear, intersecting, initial velocity patterns for the
*This investigation was aided by Research Grant GB-29097 donor. However, in practice, especially when accessible sub-
from the National Science Foundation. strate concentration ranges are limited, it may be more practical
1 In the older kinetic literature this type of behavior has been to determine the order of substrate entry into the single dis-
called “substrate activation.” Inflections in the same direction placement cycle directly by means of product inhibition and
are also yielded by allosteric systems showing “negative cooper-
ativity.” dead-end inhibition studies in the high range of acceptor con-
* All of the formal mechanisms and corresponding rate equa- centration, as Pabst et al. have done (1). The order of substrate
tions presented here are given in their simplest forms, omitting entry into a double displacement cycle is generally obvious.
all nonessential complexes. Inclusion of kinetically significant
binary and ternary complexes would not alter the plot forms or BRIDGED FORMS
the conclusions reached. In all of the schemes, A and B are
substrates; X, Y, and 2 are products; E is the free enzyme; and Folk and his collaborators (3, 4) have established as operative
E’ and E” are substituted enzymes. The same symbols represent with transglutaminase a formal mechanism that resembles a
the corresponding concentrations in the rate equations. double displacement with a “bridge” across the middle (Scheme
 Glutathione S-Transferase A: A NOVEL KINETIC MECHANISM IN WHICH
THE MAJOR REACTION PATHWAY DEPENDS ON SUBSTRATE
CONCENTRATION
Michael J. Pabst, William H. Habig and William B. Jakoby
J. Biol. Chem. 1974, 249:7140-7148.

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