From the
Mutant human
Szewczuk and Connell
(15) suggested that a carboxyl group and a thiol were present at
the active center of
In the present
study, we investigated the properties of mutant
The
enzymes were detected by enzyme activity assay at each chromatographic
step for the wild-type and the partially active mutants. An
immunological method was used to detect the enzyme fractions of mutants
that had very low levels of activity. Fifty µl of each fraction
obtained in the chromatography was added to a well of a 96-well
microtiter plate (Nunc), and the plate was incubated at room
temperature for 1 h to allow attachment of the proteins to the wells.
After blocking with 1% bovine serum albumin (in PBS), and washing the
plate with Dulbecco's phosphate-buffered saline containing 0.05%
Tween 20 (T-PBS) three times, 50 µl of goat anti-human kidney
The mutants listed in were expressed in the
baculovirus system as described under ``Experimental
Procedures.'' Several of these (D390A, D559N, and D559E) were
successfully expressed, as indicated by determinations of enzymatic
activity of the crude cell extracts. Their activities were 26, 6.0, and
7.0 milliunits/mg cell protein, respectively, whereas activity in
noninfected cells as a control was nearly undetectable (0.015
milliunits/mg cell protein). However, although the cell extracts
exhibited activity, the corresponding proteins could not be
significantly detected by the enzyme-linked immunosorbent assay system
(which did not detect less than 1% of the protein formed in extracts of
cells expressing the wild-type enzyme). It appears that these mutants
are expressed, but at low levels. The low activities observed in the
cell extracts appear to be related to low expression level rather than
to expression of proteins that have intrinsically low enzymatic
activities. Consistent with this interpretation is the finding that
mutant D390E (in contrast to mutant D390A, which was very poorly
expressed), was significantly expressed (6.6% of wild-type protein
expression and 7.3% of wild-type enzyme activity).
The wild-type
enzyme and the mutant enzymes that were expressed in sufficient amounts
were purified. Each of the purified enzymes was found to exhibit two
bands on SDS-polyacrylamide gel electrophoresis that corresponded to
M
On-line formulae not verified for accuracy
The catalytic
properties of D390E and of C454A were similar to those of the wild type
(Tables II and III). The two substitutions made for Asp-422 led to
decreased V
Determinations of the kinetic parameters for
hydrolysis () illustrate the expected decrease in
V
Although replacement of Asp-423 leads to decrease in
V
These studies constitute a further effort to identify the
functional amino acid residues at the active center of this enzyme,
whose three-dimensional structure is still unknown. Such information
will be valuable for interpretation of crystallographic studies, some
of which are now in progress, on the
The studies
reported here indicate that, in contrast to Cys-454, Asp-423 is an
important active site residue.
-glutamyl transpeptidases with amino acid
substitutions on the light subunit at the Asp residues conserved among
several species, and at the unique cysteine residue (Cys-454), were
prepared and expressed in a baculovirus insect cell system. Replacement
of Asp-423 by Ala or Glu led to major loss of enzyme activity,
consistent with the conclusion that Asp-423 is essential for activity.
A mutant in which Cys-454 was replaced by Ala was fully active,
indicating that the unique light subunit thiol is not required for
catalysis. Kinetic analysis of the hydrolysis reaction of
L-
-glutamyl-p-nitroanilide indicated that the
decreased activity of Asp-423 mutants is the consequence of an
extremely high substrate K
value, which
is more than a 1000-fold greater than that for the wild-type enzyme,
whereas the V
is decreased only less than
90-fold. The results suggest that Asp-423, and to a lesser extent
Asp-422, interact electrostatically with the
-amino group of the
-glutamyl donor substrate. Although further studies are required
to evaluate the possibility that the reaction involves function of a
charge (or proton) relay system, the present work suggests that the
-glutamyl moiety of the substrate binds electrostatically to
specific groups on the enzyme; this facilitates
-glutamyl enzyme
formation.
-Glutamyl transpeptidase is a membrane-bound heterodimeric
glycoprotein that functions in glutathione
metabolism
(1, 2, 3, 4, 5) . The
enzyme catalyzes transfer of the
-glutamyl moiety from
-glutamyl compounds as well as from glutathione to a number of
amino acids and certain dipeptides. Transfer of the
-glutamyl
moiety to water leads to hydrolysis. It is thought that a
-glutamyl enzyme intermediate, in which the
-glutamyl group
is covalently bound to the enzyme, is involved in the catalytic
mechanism. Since formation of such an intermediate is analogous to the
acyl enzyme formed by certain proteases, serine or cysteine residues
have been considered as possible active site
residues
(6, 7, 8, 9) . It has been
suggested that the catalytic site is located on the light
subunit
(6, 10) , which is consistent with the finding
that the separated light subunit exhibits protease activity (11). It
has not been examined directly whether the unique cysteine residue of
the light subunit is required for catalysis. There is accumulating
evidence suggesting that a hydroxyl group is involved in
catalysis
(7, 9, 12) . Thr-523 in the rat enzyme
has been identified as the residue that binds the potent inhibitor
acivicin
(12) . Acivicin binds to Ser-405 in the pig enzyme, and
to Ser-406 in the human enzyme (13, 14).
(
)
Despite efforts to elucidate the chemical basis of the
reaction mechanism of
-glutamyl transpeptidase, it is still
uncertain as to which enzyme group acts as a nucleophile against the
carbonyl moiety of the
-glutamyl bond.
-glutamyl transpeptidase; Elce (16)
emphasized the importance of a carboxyl group and an amino group in the
catalytic mechanism. The specific acidic amino acid residue(s),
however, have not yet been identified. An Asp residue might constitute
part of a charge (or proton) relay system (catalytic
triad)
(17) . Alternatively, the negative charge of the acidic
amino acid residue might function to attract the positive charge
(
-amino group) of the substrate. Such an important residue is
often strictly conserved among different species.
-glutamyl
transpeptidases in which conserved Asp residues on the light subunit
are replaced. We also examined directly whether the sole thiol on the
light subunit is required for catalysis by preparing a mutant enzyme in
which this Cys residue is replaced by Ala.
Materials
Restriction endonucleases and DNA
modifying enzymes were obtained from New England BioLabs.
Oligonucleotide primers were synthesized by Integrated DNA
Technologies, Inc. L--Glutamyl-p-nitroanilide,
glycylglycine, and other common reagents were purchased from Sigma.
Construction of the Transfer Plasmid
An
NcoI-EcoRI 1.8-kilobase pair DNA fragment containing
the entire coding region of human -glutamyl transpeptidase
(8) was excised from the expression plasmid used
previously
(18) . The fragment was then ligated to the
pBluescript SK+ (Stratagene) in which an NcoI site had
been created at the SmaI site of the vector by insertion of an
NcoI linker. The NotI-EcoRI fragment
prepared from that plasmid was inserted into a pVL1392 transfer vector
(Invitrogen). The resulting plasmid was used to generate the
recombinant baculovirus.
Site-directed Mutagenesis
Site-directed
mutagenesis was carried out using synthetic oligonucleotide primers
according to Kunkel (19). The uracil-substituted single stranded DNAs
were prepared from Escherichia coli CJ236 transformed by
pBluescript KS+ containing a HindIII-BamHI
1.2-kilobase pair fragment at the 5`-coding region or a
BamHI-EcoRI 0.8-kilobase pair fragment encoding the
3`-region of the cDNA for human -glutamyl transpeptidase. The
uracil templates were used with oligonucleotide primers to generate
mutant sequences. The oligonucleotide primers used in this study are
given in . Conserved Asp residues of the light subunit were
chosen on the basis of sequence identity among
rat
(8, 20, 21) ,
human
(8, 22, 23) , pig
(24) and E.
coli(25)
-glutamyl transpeptidases, and human
-glutamyl transpeptidase-related enzyme
(26) . Asp-422 is
conserved in all but the last of these; Asp-423 is conserved in all of
these. The mutations obtained were confirmed by dideoxy
sequencing
(27) , as were the entire sequences after mutagenesis.
The corresponding regions of wild-type cDNA were replaced by the mutant
sequences. The transfer plasmids for the mutant enzymes were prepared
similarly to that for the wild-type enzyme and used for transfection.
Cell Culture and General Manipulation of
Viruses
Spodoptera frugiperda (Sf) 21 cells were
maintained at 27 °C in Grace's insect media (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum, 3.33
g/liter yeastolate, 3.33 g/liter lactalbumin hydrolysate, and 50
mg/liter gentamicin. Recombinant viruses were manipulated as
described
(28) .
Preparation of Recombinant Viruses
The purified
transfer plasmids containing wild-type or mutant -glutamyl
transpeptidase cDNAs (1 µg) were co-transfected into 5
10
Sf21 cells with 10 ng of Auto-grapha californica nuclear polyhedrosis viral DNA (BaculoGold DNA, PharMingen).
Transfection experiments were carried out using the Lipofectin (Life
Technologies, Inc.) method
(29) . Media containing the generated
recombinant viruses were collected 5 days after transfection. The
recombinant viruses were further amplified to more than 5
10
plaque-forming units/ml.
Expression of Recombinant
2 -Glutamyl Transpeptidases
in Insect Cells
(30
10
Sf21 cells
were infected with the recombinant viruses carrying either wild-type or
mutant
-glutamyl transpeptidases at multiplicity of infection of
4. The infected cells were harvested about 90 h post-infection.
Purification of the Recombinant Enzymes
Sf21 cells
producing the recombinant enzymes were pelleted by centrifugation at
4000 g for 20 min. The cells were homogenized in 10
mM sodium phosphate buffer (pH 7.0) with a glass-glass
homogenizer and then stirred for 1 h. The homogenates were centrifuged
at 20,000
g for 30 min, and the pellets were
resuspended in 10 mM sodium phosphate buffer (pH 6.5). The
enzymes were extracted from the suspensions with Triton X-100 (1%) and
further solubilized by protease treatment for 5 h at room temperature;
papain (Sigma) was added at the amount of the total protein content of
the extract. The solubilized enzymes were applied on a hydroxylapatite
(Bio-Rad) column pre-equilibrated with 10 mM sodium phosphate
buffer (pH 6.5) and then eluted with a linear gradient established
between 10 mM and 0.5 M buffer. The active fractions
were further purified by isoelectric chromatography using PBE 94 and
polybuffer 74 (Pharmacia). The enzymes eluted from PBE 94 by use of a
pH gradient from pH 7.4 to pH 4.0 were passed through a column of
Sephacryl S-200 HR (Pharmacia) equilibrated with phosphate-buffered
saline (PBS)
(
)
to remove polybuffer.
-glutamyl transpeptidase antibody
(31) diluted with T-PBS
was added to each well. The plate was incubated and washed as described
above. Horseradish peroxidase-conjugated rabbit anti-goat IgG antibody
(Sigma) was then used as a second antibody. After washing the plate,
colors were developed by use of the peroxidase-substrate kit (Bio-Rad),
according to the manufacturer's instructions.
Enzyme Immunoassay
Enzyme-linked immunosorbent
assay for human -glutamyl transpeptidase was used to estimate the
relative expression levels of
-glutamyl transpeptidase proteins
produced in the insect cells. The assay and analyses were carried out
basically as described
(18) , using a peroxidase-substrate kit
(Bio-Rad) to develop the color.
Electrophoresis
The purified enzymes were
subjected to SDS-polyacrylamide gel electrophoresis analysis on 11%
gels, according to Laemmli
(32) . The proteins were visualized by
ammonical silver staining
(33) .
Enzyme Activity Assay
Standard assay for
-glutamyl transpeptidase activity was performed at 37 °C using
1 mML-
-glutamyl-p-nitroanilide as a
donor substrate and 20 mM glycylglycine as an acceptor in 0.1
M Tris-HCl buffer (pH 8.0) as described
(2) . One unit
of activity is defined as the quantity of enzyme that releases 1
µmol of p-nitroaniline/min.
Kinetic Analysis
Enzymatic activity for
transpeptidation was assayed at 37 °C using 0.25-2
mML--glutamyl-p-nitroanilide and
5-80 mM glycylglycine, as donor and acceptor substrates,
respectively, in 0.1 M Tris-HCl buffer (pH 8.0). In assessment
of hydrolysis, 6.7 µM to 5 mML-
-glutamyl-p-nitroanilide was used in the
absence of glycylglycine. Kinetic parameters for hydrolysis were
calculated using the data of the range in which a substrate activation
kinetics did not appear. When the wild-type and mutant enzymes, except
Asp-423-substituted mutants, were assayed, 0.02-0.05 µg of
the purified enzymes was subjected to each activity determination;
2-10 µg of purified enzymes were used for Asp-423 mutants.
The release of p-nitroaniline was monitored at 410 nm using a
Cary model 210 spectrophotometer (Varian). In some studies on mutants
whose activities were too low to permit continuous monitoring, the
release of p-nitroaniline was assessed by end point assay. The
reaction rates by enzymes were determined after subtraction of the
contribution of the spontaneous hydrolysis of the substrate without an
enzyme. Kinetic parameters were calculated using nonlinear regression
analysis based on the Marquart algorithm. When transpeptidation,
facilitated by glycylglycine as an acceptor substrate, was examined in
some mutant enzymes as well as the wild-type, 50 µML-
-glutamyl-p-nitroanilide was used as a donor
substrate without or with 0.25, 0.5, 1.0, 2.0, and 4.0 mM
glycylglycine as an acceptor in 0.1 M Tris-HCl buffer (pH
8.0).
Protein Determination
Protein contents were
determined by bicinchoninic acid method using bovine serum albumin as
the standard (34).
values of 44,000 and 24,000, respectively, for
the heavy and light subunits of the enzymes. The specific enzymatic
activities of these () were determined by the standard
assay (with L-
-glutamyl-p-nitroanilide and
glycylglycine). Replacement of Asp-390 by Glu, and of Cys-454 by Ala,
did not lead to decrease of specific activity. Replacement of Asp-422
by Ala or Glu did not lead to major loss of enzymatic activity. In
contrast, the mutants in which Asp-423 was replaced by Ala or Glu, and
the double mutant (D422E + D423E), exhibited very low enzymatic
activities; thus, mutant D423A showed only about 0.001% of the specific
activity of the wild-type enzyme. When the mutants in which Asp-423 was
replaced were assayed in the presence of glycylglycine, there was no
enhancement of the rate of p-nitroaniline formation; such
enhancement was found, as expected, with the wild-type enzyme (data not
shown). At concentration range higher than 80 µML-
-glutamyl-p-nitroanilide in the absence of
glycylglycine, the wild-type enzyme exhibited a substrate activation
kinetics clearly, which does not give a linear curve on the
double-reciprocal plot
(35) (Fig. 1).
L-
-Glutamyl-p-nitroanilide serves as an acceptor
substrate as well as a donor, and autotranspeptidation reaction
proceeds much more rapidly than hydrolysis. Nevertheless, this type of
kinetics was not observed in Asp-423-substituted mutants, unlike the
case of the wild type (Fig. 1). These results showed that these
mutants lacked transpeptidation reaction and only catalyze hydrolysis.
Figure 1:
Double-reciprocal plots of activity in
the absence of an acceptor substrate for the wild-type and Asp-423
mutant human -glutamyl transpeptidases. Activities were assayed at
37 °C in 0.1 M Tris-HCl buffer (pH 8.0), without an
acceptor substrate. Concentrations of
L-
-glutamyl-p-nitroanilide used were in the
range of 0.08-5 mM. The panel of the wild type also
includes an inset showing the values obtained at
concentrations less than 0.08 mM.
Kinetic parameters for transpeptidation were determined for the
enzymes purified from several mutants and from the wild type
(I), according to the following equation,
values for transpeptidation. Mutant
D422A had somewhat increased K
value for
L-
-glutamyl-p-nitroanilide and increased
K
for glycylglycine as compared with the
enzyme isolated from mutant D422E. These observations suggest that
Asp-422 may interact with the donor substrate in the
-glutamyl
binding site.
for D423A (which is significantly less than
found for D423E) and also show that the loss of a negative charge
(D422A) increases K
. On the other hand,
replacement of Asp-422 by Glu did not increase the
K
as much as did replacement by Ala.
Thus, it appears that the negative charge at Asp-422, although not
required for catalysis, may be involved in substrate binding.
, it produces a dramatic increase in
K
to values that are a 1000-fold or
higher than that of the wild type. Unlike the effect of Glu
substitution at Asp-422, substitution of Glu for Asp-423 increases
V
and K
(as
compared with the effect of Ala substitution); possibly the effect is
steric, related to the more bulky Glu moiety, which may offset the
electrostatic effect. It is interesting to note that the double Glu
mutant (D422E + D423E) has a lower K
than D423E consistent with occurrence of interaction between
neighboring carboxyl groups.
-glutamyl transpeptidase of
E. coli(36) . Amino acid residues that are thought to
be located at or near the active site include Thr-523
(rat
(12) ), Ser-405 (pig
(14) ), Ser-406
(human
(14) ), and several residues of the heavy subunit
(rat
(37) , human
(18) ). Data suggesting the participation
in catalysis of a hydroxyl group
(7, 9) , an amino
group
(16) , a carboxyl group
(15, 16) , a cysteine
residue
(8, 15, 16) , a histidine residue (4),
and an arginine residue
(18, 37, 38) have been
published. The present studies show that Cys-454 (equivalent to Cys-453
in the rat enzyme), which is the sole thiol on the enzyme's light
subunit, is not required for catalysis. Thus, the human enzyme mutant
C454A is fully active catalytically (). This cysteine
residue is conserved among the mammalian
-glutamyl transpeptidases
whose primary structures are
known
(8, 20, 21, 22, 23, 24, 39) .
Interestingly, the known bacterial enzymes have serine residues at the
corresponding positions (25, 40). It cannot be excluded that this
cysteine residue has a regulatory or other function in the mammalian
enzymes which is not required in the bacterial enzymes.
(
)
Its replacement
by Ala or Glu led to marked decreases in specific activity
(). Nevertheless, V
(hydrolysis)
values were about 1% of the wild type (), but there was
more than a 1000-fold increase in K
. Such
data do not seem to reflect operation of a charge (or proton) relay
system (Ser-His-Asp)
(17) , where one might expect a much greater
effect of amino acid replacement on
V
(41, 42, 43) . The
evidence given above suggests that Asp-422 also participates, together
with Asp-423, in substrate binding rather than in catalysis, although
Asp-423 is clearly more important. The findings suggest that these
adjacent residues (Asp-422 and Asp-423) perform essentially the same
function, i.e. to anchor the amino group of the
-glutamyl
amino acid substrate to the enzyme. It has been suggested that a
conformational change may occur after formation of the
-glutamyl
enzyme which facilitates binding of the acceptor substrate
(44) .
Such a conformational change may not be possible, or be hindered, in
the mutants that lack Asp-423, thus accounting for the observed
decrease in the transpeptidase activity exhibited by them. It seems
notable that a similar loss of transpeptidase activity was previously
found in a mutant in which Arg-107 was replaced
(18) ; this
residue is thought to interact with the carboxyl group of the
-glutamyl amino acid substrate. Accordingly it would seem that
both the
-amino group and the
-carboxyl group of the
-glutamyl donor may attach to electrostatic binding sites and that
such binding may favor orientation of the
-carboxyl moiety so that
it can interact with the
-glutamyl binding site (probably a
hydroxyl group) on the enzyme (Fig. 2).
Figure 2:
Possible role of Asp-423 in human
-glutamyl transpeptidase.
Table:
Oligonucleotide primers used for mutagenesis and
designations of mutants
Table:
Specific activity of purified -glutamyl
transpeptidases
Table:
Kinetic parameters for wild-type and mutant
-glutamyl transpeptidases in transpeptidation
Table:
Kinetic parameters
for wild-type and mutant -glutamyl transpeptidases in hydrolysis
(in 0.1 M Tris-HCl buffer (pH 8.0) at 37 °C)
-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.
-glutamyl transpeptidase showed that inactivation of
the enzyme by treatment with iodoacetamide leads to esterification of
Asp-422 (equivalent to Asp-423 in the human enzyme (45)).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.