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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
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
r 100
90
12
90
70
60
OLC
20
--
/”25
m
FRACTION
30
NUMBER
35
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
9. p-Nitrophenethyl bromide
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)
, 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
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
+ kelk3k4kl’k2’k3’AG2
E
2,
Y
SCHEME 1
7146
.8
.6 .6
4 .4
.2 .2
4 18
.8 -
C
- 4.0
.6 3.0
.2 1.0
0 0
.8 4.0
.6 3.0
012345 ‘t-f-8- 10 I2 I4 I6 I8 20
APPENDIX
DISTINCTION AMONG FORMAL MECHANISMS THAT YIELD DOUBLE RECIPROCAL PLOTS CONCAVE
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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