From the
Studies carried out after the primary structure
of the enzyme was
determined
(8, 9, 10, 11, 12) led to identification of Thr-523 of the rat enzyme as the
site of attachment of the inhibitor acivicin (13).
To ultimately understand the structure
and mechanism of action of the enzyme, more information is needed about
the nature of the active site residues and their locations. In the
present work, in which the interaction of the enzyme with iodoacetamide
and N-acetylimidazole was probed further, we have obtained
evidence for the specific amino acid residues of the enzyme that react
with these compounds. We also found that the inactivation of the enzyme
that occurs when it is incubated with iodoacetamide can be reversed by
treatment with hydroxylamine. We have observed a stabilized
N-Acetylimidazole was prepared according to the method of
Reddy et al.(18) ;
N-L[-
Fisher was the source of boric acid and dialysis tubing (molecular
weight cut off 6-8 kDa) and Mallinckrodt for hydroxylamine and
guanidinium hydrochloride from Schwarz-Mann. Pierce Chemical Co.
supplied the constant boiling 6 M HCl, amino acid standards,
and phenylisothiocyanate that were used for Pico-Tag amino acid
analysis. National Diagnostics was the source of Monofluor
scintillation liquid, and S-carboxymethyl-L-cysteine
was obtained from Chemical Dynamics Corp. Low molecular weight
standards for gel-electrophoresis and protein assay dye were purchased
from Bio-Rad.
Dialysis using tubing having a molecular weight cutoff
of 6-8 kDa was carried out to remove excess modifying agent when
necessary, against three changes of 4 liters each of Tris-HCl (5
mM, pH 8) buffer, for 36-48 h at 4 °C. The presence
of both L-serine and borate (S/B), each at a final
concentration of 50 mM, which acts as a competitive
inhibitor
(21, 22) , was used to protect the active site
of the enzyme. Solid iodoacetamide and iodoacetate (in the presence of
maleate (50 mM)) were added as indicated to a final
concentration of 100 mM; the pH values of the solutions were
readjusted to 8 with NaOH or HCl (0.1 M). After 6 h at 37
°C, excess 2-mercaptoethanol was added for 30 min before dialysis.
N-Acetylimidazole was added as a solid to a final
concentration of 100 mM and incubated for at least 12 h; the
pH values of these solutions were not readjusted; the final values were
usually 7.8.
In the experiments described in , the final
volumes were brought to 4 ml by speed vacuum lyophilization. Each
experiment was then split into four equal parts (
The bound radioactivity was determined by counting the
radioactivity in 10 µl (10%) of the solution. To ascertain to which
subunit the radiolabel was bound, 25 µl (25%) of the solutions were
subjected to gel-electrophoresis as described above. SDS-polyacrylamide
gel electrophoresis of the enzyme showed two bands corresponding to
molecular masses of
The remainder of the solutions (65 µl)
were treated with guanidinium hydrochloride (8 M) and acetic
acid (1 M) at 37 °C for 18 h. (final volume, 250 µl).
The subunits were separated by reversed-phase HPLC on a Vydac C
Separation of the peptides generated was conducted on
a Waters system with a µBondapak C
The protein was precipitated after addition of bovine serum albumin
(1 mg), by adding trichloroacetic acid to a final concentration of 20%.
After incubation at 0 °C for 30 min, the samples were centrifuged
in a Beckman Microfuge B for 5 min at 4 °C. The supernatant was
removed carefully, and the precipitated protein was washed twice with
100-µl portions of 5% trichloroacetic acid. Portions of the
precipitated protein and of the combined washings were analyzed for
radioactivity. The remainder of the precipitated protein was subjected
to gel-electrophoresis to separate the subunits, whose content of
radioactivity was determined.
In the present studies we made the interesting
observation that the iodoacetamide-inhibited enzyme can be reactivated;
thus, when the iodoacetamide-inhibited enzyme was incubated with
hydroxylamine (200 mM, pH 8) at 37 °C, about 75-80%
of the initial activity was restored after 12 h at 37 °C. Only
slight (<5%) reactivation was observed when the inactivated enzyme
was incubated in sodium phosphate buffer (50 M, pH 8) in the
presence or absence of dithiothreitol (25 mM), glycylglycine
(0.1 M), or GSH (0.2 M).
Incubation of the enzyme
with N-acetylimidazole also led to inactivation. Serine and
borate did not protect against inactivation. The activity was not
restored by incubation of the inactivated enzyme with hydroxylamine,
dithiothreitol, or substrates.
Addition of acivicin to the
S/B-protected enzyme (experiment 3) resulted in the same degree of
labeling as the control (experiment 1). Treatment with
N-acetylimidazole (experiment 10) shows that about eight
residues (about three on the heavy subunit and about five on the light
subunit) are not modified by iodo[
To identify the residues modified by
iodo[
The
Modification of the enzyme with
N-[1-
Other evidence
indicating formation of a stabilized
The stability of the
The present studies indicate that the active site carboxyl
group of
Another amino acid residue at the
active site (cysteine) was exposed by treatment of the enzyme with
N-acetylimidazole so that it became available for reaction
with iodoacetamide (cf. Ref. 6). This is the only cysteine
residue on the light subunit and is therefore identified as Cys-453.
Although it is located in the active site region, this thiol is
unreactive in the native enzyme in which it is apparently
``buried'' (see Ref. 28).
The data indicate that about
five amino acid residues are acetylated by treatment of the enzyme with
N-acetylimidazole and that residues of both subunits are
acetylated. It seems significant that one of these is Lys-99, which
previous studies
(16) suggested is at or near the binding site
for acceptor substrates. Interestingly, Lys-99 was found to be modified
by phenylglyoxal
(16) , which is generally regarded as an
arginine reagent. Acetylation evidently affects one or more of the
amino acid residues needed for catalysis; since it stabilizes the
The
In
summary, the present studies have identified two active site residues
of rat
See ``Methods'' for experimental details. After
completion of the chemical treatments indicated, the samples were
incubated for 24 h at 37 °C with
iodo[2-
Each of the experiments was carried out in
sequential order as described under ``Methods.''
[3-
The
chemically modified enzymes were incubated with
[
We thank Dr. Einar Stole and Dr. Yoshitaka Ikeda for
very helpful discussions and Edith Perryman, Selma Haschemeyer, and
Linda Gilbert for their assistance in preparing this manuscript for
publication.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Glutamyl transpeptidase, an enzyme of central significance
in glutathione metabolism, is inactivated by iodoacetamide, which
esterifies an active site carboxyl group identified here as that of
Asp-422. Treatment of the inactivated enzyme with hydroxylamine leads
to de-esterification and to restoration of enzymatic activity.
N-Acetylimidazole, which also inactivates the enzyme,
acetylates several amino acid residues. Acetylation exposes Cys-453,
which is buried in the native enzyme, to reaction with iodoacetamide.
Incubation of the acetylated enzyme with glutamine produces a
stabilized
-glutamyl-enzyme form which is (a) located
exclusively on the light subunit, (b) more labile to base than
to acid, (c) destabilized by denaturation of the enzyme with
guanidinium ions, and (d) reactive with hydroxylamine to form
-glutamylhydroxamate. Stabilization of the
-glutamyl-enzyme
appears to be associated with acetylation of lysine residues (including
Lys-99). These and other findings suggest that the
-amino group of
the
-glutamyl substrate is linked electrostatically to Asp-422 so
as to facilitate reaction of the
-carbonyl of the substrate with
an enzyme hydroxyl group to form a
-glutamyl-enzyme.
-Glutamyl transpeptidase is a glycoprotein consisting of
two nonidentical
subunits
(1, 2, 3, 4, 5) . The
light subunit is bound to the heavy subunit by noncovalent
interactions; the heavy subunit has a hydrophobic N terminus that
anchors the enzyme to the extracellular membrane. The enzyme catalyzes
cleavage of the
-glutamyl bond of glutathione and related
compounds, to form a postulated
-glutamyl-enzyme intermediate,
which may be cleaved by water to form glutamate (hydrolysis), or the
-glutamyl moiety may be transferred to acceptors such as amino
acids (transpeptidation). Definitive evidence for formation of a
-glutamyl-enzyme intermediate is still needed. It has been
suggested that the
-glutamyl moiety attaches to an enzyme hydroxyl
group
(1) , but other possibilities exist; studies on the nature
of this bond led to the conclusion that the
-glutamyl group is
attached by amide linkage to an enzyme amino group
(6) . Szewczuk
and Connell
(7) found that the enzyme was irreversibly inhibited
by treatment with iodoacetamide, that this inhibition was prevented by
a mixture of serine and borate, and that acid hydrolysis of the
inhibited enzyme led to formation of glycollic acid, which was
postulated to be derived from an alkylated group at the active center
of the enzyme. Elce
(6) found that treatment of the enzyme with
N-acetylimidazole, followed by treatment with
iodo[
C]acetamide led to labeling of two amino
acid residues at the active site; one has a carboxyl group and the
other is a cysteine.
(
)
Although previous studies suggested (1, 14, 15) that the
active site is situated on the light subunit, selective labeling
experiments on the rat enzyme with
[
C]phenylglyoxal showed labeling of Lys-99 and
Arg-111 (on the heavy subunit)
(16) , and site-specific
mutagenesis studies of the heavy subunit of the human enzyme suggested
that Arg-107 plays a significant role in binding of
-glutamyl
substrate and that Glu-108 participates in acceptor substrate binding
and catalysis
(17) .
-glutamyl enzyme form that exhibits properties that are consistent
with this postulated intermediate in the reactions catalyzed by the
enzyme.
Materials
-Glutamyl transpeptidase was isolated (specific
activity, 1100 units/mg) from frozen rat kidneys (Pel-Freez
Biologicals) and solubilized by papain digestion
(1) .
Endoproteinase Glu-C (EC 3.4.21.19) and endoproteinase Lys-C (EC
3.4.21.50) were purchased from Boehringer Mannheim. Other chemicals
were purchased from Sigma except as noted.
L-(
S,5S)-
-Amino-3-chloro-4,5-dihydro-5-[3-
C]isoxazole
acetic acid (acivicin, AT-125) with specific radioactivity of 31
mCi/mmol and unlabeled acivicin were kindly supplied by Dr. R. S. Hsi
of the Upjohn Company. Iodo[2-
C]acetic acid and
iodo[2-
C]acetamide (specific radioactivity, 55
mCi/mmol) and L-[U-
C]glutamine
(specific radioactivity, 270 mCi/mmol) were obtained from Amersham
Corp. HPLC
(
)
solvents were obtained from VWR
Scientific Corp. Imidazole and methylamine was obtained from Eastman
Kodak Co., and acetic anhydride was purchased from Aldrich.
C]acetylimidazole was
prepared by slight modification, as follows.
[1-
C]Acetic acid (100 µCi; specific
radioactivity, 12.4 mCi/mmol) was added to acetic anhydride (200
µl; 3.2 mmol) and stirred for 5 min; with further stirring,
imidazole (108.8 mg, 1.5 mmol) (recrystallized thrice from ethyl
acetate) was added, and the mixture was placed for 1 h at 20 °C.
Excess L-[
C]acetic anhydride was
removed by vacuum distillation, and the remaining white solid was
crystallized from ethyl acetate. The resulting
N-[1-
C]acetylimidazole (101 mg, 1.5
mmol, 31 µCi/mmol) was stored in a desiccator at -20 °C.
Methods
Gel Electrophoresis
The gels were 10%
SDS-polyacrylamide, prepared according to Weber and Osborn
(19) and run in duplicate on a Mighty Small II (SE 250)
gel-electrophoresis apparatus (Hoefer Scientific) at a voltage of 100 V
for 1 to 1.5 h. Estimates of the molecular weights of the subunits were
made using low molecular weight standards; staining was done with
Coomassie Blue R-250. The lanes of identical gels were cut out and each
was cut into five equal parts, which were shaken with 0.5 ml of
Tris-HCl (0.1 M, pH 8) containing 0.1% SDS for 24 h. Liquid
scintillation fluid (4 ml) was added, and the radioactivity was
determined with a LKB 1218 RackBeta liquid scintillation counter.
Enzyme Activity Assay
Standard -glutamyl
transpeptidase activity assays were performed with
L-
-glutamyl-p-nitroanilide (1 mM) and
glycylglycine (20 mM) in Tris-HCl (0.1 M, pH 8) at 37
°C
(20) . The rate of release of p-nitroaniline was
monitored at 410 nm (
= 8800 M
cm
) (Varian Cary 219 spectrometer). Portions
(5-10 µl) taken at appropriate times after chemical
modification and reactivation were assayed (1 unit is equivalent to the
release of 1 µmol of p-nitroaniline/min.).
Inactivation Studies
The enzyme (125 units)
was incubated at 37 °C in a solution (100 µl) of Tris-HCl (0.1
M, pH 8), containing the various inhibitors and other
compounds. At intervals, portions (5 µl) were assayed for activity.
Chemical Modification Experiments
The chemical
modifications were carried out at 37 °C in Tris-HCl (0.1
M, pH 8) with between 550 and 600 units of enzyme per
experiment.
1 ml each), and
one of the following was added to each; (a)
iodo[2-
C]acetamide (2.75 mCi/mmol; final
concentration, 100 mM); (b)
iodo[2-
C]acetic acid (2.75 mCi/mmol; final
concentration, 100 mM), in the presence of maleic acid (50
mM); (c) [3-
C]acivicin (3.1
mCi/mmol; final concentration, 1 mM); and (d)
L-[U-
C]glutamine (5.4 mCi/mmol; final
concentration, 1 mM). These solutions were then incubated at
37 °C for 24 h, and the radiolabeled enzymes were separated from
excess radioactive compounds by rapid gel filtration using a Sephadex
G-25 (fine) column (0.5
5 cm) equilibrated with sodium
phosphate (5 mM, pH 8)
(23) . The pooled fractions were
concentrated to 100 µl, and the following analyses were carried
out.
42 and
22 kDa for the heavy and light
subunits, respectively.
column (0.46
25 cm) using a Waters system and 0.1%
trifluoroacetic acid as solvent A and 95% (v/v) acetonitrile/0.1%
trifluoroacetic acid as solvent B. A linear gradient from 20 to 60%
solvent B for 40 min was used with a flow rate of 1.5 ml/min. The
effluent was monitored at 214 nm, and fractions were collected each
minute; the separated subunit fractions were pooled and concentrated.
Portions of the eluted subunits were taken for liquid scintillation
counting.
Amino Acid Analysis
The enzyme preparations were
hydrolyzed with 6 M HCl at 110 °C under N for
20-24 h. The hydrolysates were neutralized, and the amino acids
present were converted to the corresponding phenylthiocarbamyl amino
acids and analyses were performed as described by Bidlingmeyer et
al.(24) . Fractions (1 min) of the eluent were collected
for liquid scintillation counting.
Proteolytic Digestion
Several chemically modified
enzyme samples were subjected to proteolytic digestion at 37 °C for
24 h using endoproteinase Glu-C (10% by weight) in
NaHPO
(50 mM, pH 7.8) or
endoproteinase Lys-C (10% by weight) in Tris (100 mM, pH 8.2)
containing urea (2 M) and methylamine (20 mM).
Digestion with
-chymotrypsin (5% by weight) was carried out for 6
h in Tris-HCl (50 mM, pH 7.8) containing CaCl
(5
mM).
ODS (3.9 mm
30 cm) using trifluoroacetic acid (0.1%) as solvent A and 95% (v/v)
acetonitrile (0.1% trifluoroacetic acid) as solvent B. A flow rate of
0.7 ml/min. was used in conjunction with a linear gradient between 100%
A and 60% B after 60 min and, thereafter, 100% B for 10 min. Elution of
the peptides was monitored at 214 nm. Fractions (1 min) of the eluent
were collected and assayed for radioactivity. Analysis of [
C]Inhibitor-Enzyme Linkage
Stability-Several chemical modification experiments were carried
out to investigate the stability of the
[
C]inhibitor-enzyme linkage under various
conditions; hydroxylamine (200 mM, pH 8), dithiothreitol (25
mM), guanidinium ion (8 M, pH 8), and combinations
thereof, in sodium phosphate (50 mM, pH 8), as well as in NaOH
(0.1 M) and HCl (0.1 M). The solutions (total volume,
100 µl) were incubated at 37 °C for 24 h. A 10-µl portion
(10%) was assayed for enzyme activity and compared with the controls.
Protein Determination
Bovine serum albumin was
used as a standard for protein concentration estimations using the
Bio-Rad protein assay dye
(25) . Chemical Modification of the Enzyme by
N-[1-C]Acetylimidazole-N-[1-
C]Acetylimidazole
(0.5 mmol; specific radioactivity, 31 µCi/mmol) was added to the
enzyme (4 mg,
58 nmol) in 200 µl of Tris-HCl (0.1 M,
pH 8) and incubated at 37 °C for 12 h. The excess
[
C]reagent was removed by use of a Penefsky
column
(23) ; the
C-acetylated enzyme was
lyophilized and redissolved in 2 ml of sodium phosphate (50
mM, pH 8). Analyses of the
[
C]acetyl-enzyme linkages were made under
various conditions as described above.
Inhibition of the Enzyme by Iodoacetamide and by
N-Acetylimidazole
Incubation of the enzyme with 50 mM
iodoacetamide at pH 8 led to 50% inactivation after 30 min and to
complete inactivation after 5 h. Iodoacetate (50 mM) had
little effect on activity, but in the presence of 50 mM
maleate, about 50% of the activity disappeared within 90 min. Maleate
also increased inhibition by iodoacetamide; under these conditions,
maleate itself had no effect on activity. Maleate enhances hydrolysis
of a -glutamyl donor and decreases transpeptidation, apparently by
binding to the acceptor site (26). Inactivation of the enzyme by
iodoacetamide (and by iodoacetate) was prevented by addition of
L-serine plus sodium borate (50 mM each), but not by
adding either component alone. These findings are in general agreement
with earlier work.
Modification of the Enzyme by Treatment with
Iodoacetamide, Iodoacetate, and N-Acetylimidazole
summarizes studies on the effects of iodoacetamide and
N-acetylimidazole on labeling with
iodo[C]acetamide. In these studies, samples of
the enzyme (550-600 units) were treated with serine plus borate
(S/B), acivicin (1 mM), iodoacetamide (unlabeled),
N-acetylimidazole (unlabeled), and dialyzed as indicated; then
the treated enzyme samples were incubated with labeled iodoacetamide as
stated in . (Since the results with iodoacetate (50
mM) plus maleate (50 mM) were very similar to those
found with iodoacetamide, these data are not shown.) Experiment 1 shows
that treatment of the enzyme with
iodo[
C]acetamide led to labeling of both
subunits; protection with S/B led to decreased labeling of the light
subunit equivalent to about one residue (experiment 2). Experiment 4
shows that protection of the active site by S/B, followed by treatment
with unlabeled iodoacetamide, dialysis, and treatment with
iodo[
C]acetamide, led to labeling of about one
residue of the light subunit. In experiment 9, the enzyme was treated
with N-acetylimidazole (after iodoacetamide treatment in the
presence of S/B); here, treatment with
iodo[
C]acetamide led to labeling of about two
residues of the light subunit. A similar result, labeling of two
residues, was obtained in experiment 12. In experiment 11, S/B
protected about two residues of the N-acetylimidazole-treated
enzyme from reaction with iodo[
C]acetamide.
Notably, labeling equivalent to only about one residue was found in
experiment 8 in which the unprotected enzyme was treated with
iodoacetamide prior to treatment with N-acetylimidazole and
iodo[
C]acetamide.
C]acetamide as
compared with the control. Protection of the active site with S/B
(experiments 5 and 10) does not alter the number of residues modified
by N-acetylimidazole, consistent with the finding that S/B
does not protect the enzyme against inactivation by
N-acetylimidazole.
C]acetamide, portions of the reaction
mixtures of these experiments were subjected to hydrolysis with 6
M HCl for 20-24 h at 110 °C; and derivatization and
amino acid analysis (see ``Methods'') were carried out.
S-[
C]Carboxymethylcysteine was found
only in experiments 8, 9, and 12; 0.96 ± 0.09 (n = 9) mol/mol of enzyme was found, and an additional
quantity (0.91 ± 0.11 (n = 9) mol/mol) of
C equivalent appeared in the fractions collected from
these experiments. This
C material was not a
phenylisothiocyanate derivative, but was apparently
[
C]glycolate, formed by hydrolysis of the
corresponding aspartate
-ester (see Ref. 7 and below). The labeled
enzyme obtained in experiment 4 was subjected to enzymatic digestion by
endoproteinase Glu-C; separation of the peptides obtained by
chromatography on a C
column gave a single labeled
component (retention time, 11 min), which was presumably the aspartate
-ester. Amino acid analysis after acid hydrolysis of this
component showed aspartate to be the only derivatized residue. Under
the conditions used here, endoproteinase Glu-C digestion cleaves the
carboxylate peptide bonds of both aspartate and glutamate residues, so
that the resulting
-aspartyl-[
C]glycollamide ester could only
arise from Glu-Asp or Asp-Asp. These sequences occur throughout the
enzyme, but are found only twice in the light subunit; 388,389:ED and
421,422:DD. A similar digestion of the labeled enzyme obtained in
experiment 4 (Table l) was carried out with endoproteinase Lys-C, and a
labeled peptide was obtained; the amino acid composition of this
peptide corresponded to residues 407-443 of the light subunit
(data not shown) (and not to residues 381-406), thus indicating
that the modified aspartyl is Asp-422.
C-labeled
enzyme obtained in experiment 4 () was treated with various
reagents (such as phosphate buffer, pH 8, 8 M guanidinium
hydrochloride, hydroxylamine; see ``Methods''). Under these
conditions, a large fraction (73-84%) of the radioactivity was
released on treatment with 0.2 M hydroxylamine or 0.1
M sodium hydroxide; about 30% was released on treatment with
0.1 M hydrochloric acid. Treatment with dithiothreitol,
substrate, or guanidinium ions released less than 8% of the
radioactivity. These findings are consistent with an aspartate
-ester.
C]acetylimidazole led to >99%
inhibition and binding of 4.89 ± 0.21 (n = 3)
mol of [
C]acetyl/mol of the enzyme, (i.e. modification of
five residues). Treatment of the
C-acetylated enzyme under various conditions (see
``Methods'' and Ref. 27) showed that about of
20% of the
label, equivalent to about one molar residue, was released by
dithiothreitol and
40% (equal to about two molar residues) was
released by treatment with hydroxylamine, consistent with release of
[
C]acetyl from a cysteine residue and two
tyrosine residues. SDS-gel electrophoresis studies indicated that the
presumed acetylated cysteine was on the heavy subunit, whereas one each
of the tyrosine residues was located on the heavy and light subunits.
C-Acetylated enzyme, after treatment with dithiothreitol
and hydroxylamine, was still fully inhibited and contained label
equivalent to two acetyl groups; probably these are acetylated lysines;
one was attached to each subunit. This acetylated enzyme was subjected
to endoproteinase Glu-C digestion; fractionation on a C
column separated two radiolabeled peaks. Acid hydrolysis of these
revealed that the amino acid composition of one of these corresponded
to residues 66-101, which contains only one lysine; lysine 99.
This was confirmed by treatment of the peptide with
-chymotrypsin
followed by separation of the oligopeptides produced; the label was
associated with a peptide corresponding to residues 93-101. The
other labeled peak from the endoproteinase Glu-C digestion contained
two peptides which could not be separated; these were identified on the
basis of the mixed amino acid composition as
VKRAVE and
SRKGGE; both contain a lysine (Lys-495 and Lys-561).
Binding of Acivicin to the Enzyme
Chemical
modification experiments were carried out in which
[C]acivicin was added in place of the
iodo[2-
C]acetamide used in .
Virtually all of the bound radioactivity found in these experiments
() was associated with the light subunit. Treatment with
S/B of the native enzyme (; experiment 2), and of the
N-acetylimidazole-modified enzyme (experiment 4), prevented
acivicin from binding to the enzyme. In the absence of S/B, acivicin
bound to both the native enzyme (experiment 1) and the acetylated
enzyme (experiment 3) at about a 1:1 molar ratio.
[
C]Acivicin bound to enzyme that had been
pretreated with iodoacetamide (experiment 5); iodoacetamide was
previously shown to modify an aspartyl residue in the
-glutamyl
binding site (see above). When the N-acetylimidazole modified
enzyme was treated with iodoacetamide (experiment 7), both an aspartyl
and a cysteine residue at the
-glutamyl binding site were modified
(). Experiments 5 and 7 () show that acivicin
binds to the enzyme light subunit in such a way as to not be affected
by either of the modified aspartyl and cysteine residues in the
-glutamyl binding site. However, acivicin is prevented from
binding by S/B (experiment 2). Thus, it appears that the binding
domains of S/B overlap those of acivicin. Experiment 6 also shows that
iodoacetamide treated enzyme after addition of S/B can still interact
with acivicin. Apparently the S/B complex cannot protect the
-glutamyl binding site because the iodoacetamide modified aspartyl
residue interferes. Inactivation of the enzyme by acivicin does prevent
modification of the aspartyl and cysteine residues at the
-glutamyl binding site. The binding of acivicin also prevents the
formation of a stabilized
-glutamyl-enzyme form (see next
section).
Evidence for
formation of a -Glutamyl-Enzyme Formation
-glutamyl-enzyme was obtained in studies in which
chemically modified enzyme was incubated with
[
C]glutamine (I). In all of the
experiments in which binding of [
C]glutamine was
observed, the label was present only on the light subunit. In the
absence of chemical modification (experiment 1, control), there was no
binding of [
C]glutamine. After treatment of the
enzyme with N-acetylimidazole, the equivalent of about 1 mol
of [
C]glutamine was bound (experiment 2).
Treatment of the [
C]labeled enzyme with 20
mM hydroxylamine led to >90% formation of
-[
C]glutamylhydroxamate, as identified
previously (29). Protection of the active site by adding S/B
(experiment 3), or modification with iodoacetamide before or after
N-acetylimidazole treatment (experiments 4 and 5,
respectively), prevented formation of
-glutamyl-enzyme. Treatment
with S/B before iodoacetamide treatment, which prevents modification of
the aspartyl and cysteinyl residues at the
-glutamyl binding site,
did not prevent formation of
-glutamyl-enzyme (experiments 6 and
7). Treatment of the N-acetylimidazole-modified enzyme with
acivicin prevented [
C]glutamine binding
(experiment 8). Preincubation of the
N-acetylimidazole-modified enzyme with either unlabeled
glutamine or glutathione (experiment 9) led to a marked decrease of
binding of [
C]glutamine. This was not observed
on preincubation with glutamate (experiment 10).
-glutamyl-enzyme came from
enzyme assays carried out in experiments 2, 6, and 7 (I),
in which the enzyme was modified by N-acetylimidazole. In
these studies, monitoring at 410 nm for the release of
p-nitroaniline from
-glutamyl-p-nitroanilide
showed activity within the first 10 s after mixing the substrates with
enzyme. Thereafter, the activity decreased, and no further release of
p-nitroaniline was found after the initial 20 s. In a typical
experiment, the change in absorbance between time 0 and 20 s was
equivalent to the formation of 0.6 µmol of p-nitroaniline.
The amount of enzyme assayed here (500 units, 0.45 mg) would be
expected to release 6.6 nmol of p-nitroaniline, in a single
enzyme turnover. Therefore, the enzyme turns over about 90 times before
forming a stabilized
-glutamyl-enzyme.
-[
C]glutamyl-enzyme formed from the
N-acetylimidazole-modified enzyme was examined under various
conditions (see ``Methods''). In sodium phosphate (50
mM, pH 8), with or without glycylglycine (0.1 M), the
-[
C]glutamyl-enzyme was relatively stable.
Release of radioactivity increased substantially upon denaturing the
enzyme with guanidinium ions (8 M, pH 8), suggesting that this
form is stabilized by its environment on the enzyme. Relatively little
radioactivity was released on incubation with dithiothreitol. Most of
the enzyme-bound radioactivity was released on treatment with
hydroxylamine, which led to formation of
-[
C]glutamylhydroxamate. This finding, and
the observation that the linkage between the
-[
C]glutamyl moiety and the enzyme was more
susceptible to base than acid, are consistent with an ester. The
-[
C]glutamyl-enzyme was subjected to
endoproteinase Glu-C digestion. However, a peptide containing
radioactivity was not found; only free
[
C]glutamate was identified.
-glutamyl transpeptidase that reacts with iodoacetamide
is Asp-422. In this reaction the
-carboxyl group of Asp-422 is
esterified to form O&cjs0808;C-OCH
CONH
,
which is associated with loss of enzymatic activity. Treatment of the
inactivated enzyme with hydroxylamine restores most of the initial
enzymatic activity. Reactivation is associated with cleavage of the
ester linkage; the details of this reaction still need study. Asp-422
may provide an essential electrostatic binding site, probably for the
-amino group of the
-glutamyl substrate. This subject is also
considered elsewhere
(28) .
-glutamyl-enzyme, it apparently affects amino acid residues that
are involved in the breakdown of the
-glutamyl enzyme. Acetylation
is probably associated with conformational change in the enzyme; the
number of residues that can be modified by iodoacetamide decreases
after acetylation ().
-glutamyl enzyme formed
here by reaction of the acetylated enzyme with glutamine
(I) has properties expected of an intermediate that might
be formed in catalysis. It is highly reactive with hydroxylamine and is
not very stable. The conditions under which it may and may not be
formed are consistent with such an intermediate
-glutamyl enzyme.
Previously, evidence was reported for formation (by a different
procedure) of an apparent
-glutamyl enzyme form in which the
-glutamyl moiety was linked to an enzyme lysine amino
group
(6) , but the enzyme form obtained in that work was found
to be stable to performic acid and to hydroxylamine, and thus its
properties were markedly different from those found here.
-glutamyl transpeptidase: Asp-422 and Cys-453. Asp-422
appears to be required for enzyme activity, whereas Cys 453, which is
buried, is probably not.
(
)
Acetylation seems to
open and expose the active site region, which, nevertheless, retains
the reactive
-glutamyl binding site. This enzyme site, which
probably has a hydroxyl group, is not identical to the acivicin binding
sites (Ser-405, Thr-523) previously identified (13, 29). We tentatively
suggest that the
-amino group of the
-glutamyl substrate is
linked electrostatically to Asp-422 in such a manner as to facilitate
reaction of the
-carbonyl of the substrate with a specific enzyme
hydroxyl group thus forming a
-glutamyl-enzyme, which may
participate in the transpeptidation and hydrolysis reactions that are
catalyzed by the enzyme.
Table:
Labeling of the
light and heavy subunits of the enzyme by
iodo[C]acetamide after treatment of the
holoenzyme with unlabeled iodoacetamide (IA) and N-acetylimidazole
(N-Ac-I)
C]acetamide. Excess
C
reagent was removed by rapid gel filtration (23), and the
C present in the light and heavy subunits was determined
after their separation by SDS-gel electrophoresis.
Table:
Binding of acivicin
to the light subunit
C]Acivicin was incubated for 24 h at 37
°C with the modified enzymes; excess
[3-
C]acivicin was removed by use of a Penefsky
column (23). Portions were subjected to gel electrophoresis, and the
mean molar ratio of
C-compound to light subunit are given
± S.D. (n = 6) (<0.02 molar ratio of
C-compound was associated with the heavy subunit).
Table:
Formation
of a stabilized -glutamyl-enzyme on the light subunit
C]glutamine for 24 h at 37 °C as indicated
above (see ``Methods''). Excess
C-compound was
removed by gel filtration and portions of the enzyme were subjected to
gel electrophoresis. The molar ratios (
C-compound to light
subunit) are given as means ± S.D. (n = 3).
-glutamyl transpeptidases differ by one residue. Thus, Asp-422 and
Cys-453 in the rat enzyme are equivalent, respectively, to Asp-423 and
Cys-454 in the human enzyme.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.