(Received for publication, June 22, 1995)
From the
The serine residue required for catalysis of -glutamyl
transpeptidase was identified by site-specific mutagenesis of the
conserved serine residues on the basis of sequence alignment of the
light subunit of human, rat, pig and two bacterial enzymes. Recombinant
human
-glutamyl transpeptidases with replacements of these serine
residues by Ala were expressed using a baculovirus-insect cell system.
Substitutions of Ala at Ser-385, -413 or -425 yielded almost fully
active enzymes. However, substitutions of Ala at Ser-451 or -452
yielded enzymes that were only about 1% as active as the wild-type
enzyme. Further, their double mutant is only 0.002% as active as the
wild type. Kinetic analysis of transpeptidation using glycylglycine as
acceptor indicates that the V
values of Ser-451
and -452 mutants are substantially decreased (to about 3% of the wild
type); however, their K
values for L-
-glutamyl-p-nitroanilide as donor were only
increased about 5 fold compared to that of the wild type. The double
mutation of Ser-451 and -452 further decreased the V
value to only about 0.005% of the wild type, while this mutation
produced only a minor effect (2-fold increase) on the K
value for the donor. The kinetic values
for the hydrolysis reaction of L-
-glutamyl-p-nitroanilide in the mutants
followed similar trends to those for transpeptidation. The rates of
inactivation of Ser-451, -452 and their double mutant enzymes by
acivicin, a potent inhibitor, were less than 1% that of the wild-type
enzyme. The K
value of the double mutant
for L-serine as a competitive inhibitor of the
-glutamyl
group is only 9 fold increased over that of the wild type, whereas the K
for the serine-borate complex, which
acts as an inhibitory transition-state analog, was more than 1,000
times higher than for the wild-type enzyme. These results suggest that
both Ser-451 and -452 are located at the position able to interact with
the
-glutamyl group and participate in catalysis, probably as
nucleophiles or through stabilization of the transition state.
-Glutamyl transpeptidase, a heterodimeric glycoprotein
anchored to the extracellular surface of cell membrane, plays an
important role in glutathione metabolism. It catalyzes the transfer
reaction of a
-glutamyl moiety from glutathione and related
compounds to a variety of amino acids and dipeptides. The transfer of
the
-glutamyl moiety to water leads to
hydrolysis(1, 2, 3) . Both the large and the
small subunit of
-glutamyl transpeptidase are encoded by a common
messenger RNA(4, 5, 6) . The enzyme is
translated as a single chain precursor, which yields subunits
post-translationally by proteolytic
processing(7, 8, 9) .
A catalytic
nucleophile, such as is found in thiol- or serine-class proteases, is
assumed to form a covalent linkage with a -glutamyl group because
reactions catalyzed by
-glutamyl transpeptidase are thought to
proceed via a
-glutamyl-enzyme intermediate(10) . Although
-glutamyl transpeptidase from mammalian species possesses a unique
thiol on the light subunit, which has the catalytic
domain(11, 12) , this cysteine residue is not required
for catalysis(13) . On the other hand, several studies suggest
that a serine residue is involved in
catalysis(14, 15) . This is also supported by the
recent observation that modification of
-glutamyl transpeptidase
with N-acetylimidazole led to a stabilized intermediate, and permitted
the detection of a
-glutamyl enzyme in which the
-glutamyl
moiety was bound on the light subunit(16) . The nature of the
linkage between enzyme and
-glutamyl group was found to be
consistent with an ester. Thus, the nucleophile in the active site was
proposed to be a hydroxyl group, probably a serine residue on the light
subunit. However, since the stabilized
-glutamyl enzyme is
hydrolyzed upon denaturation by guanidinium ions, the catalytic residue
has not yet been identified. The detailed mechanism of the catalysis of
-glutamyl transpeptidase is still unclear because its crystal
structure is unknown. Several residues, however, have been identified
to be at or near the active
site(13, 16, 17, 19, 20) .
Acivicin (L-(S,5S)-
-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic
acid), a potent inhibitor of
-glutamyl transpeptidase, inactivates
the enzyme by its covalent attachment in or near the active
site(2, 21, 22) . By use of isotopically
labeled acivicin, amino acid residues to which the agent bound have
been identified. These are Thr-523 in rat enzyme(17) , Ser-405
in pig and Ser-406 (equivalent to Ser-405 in rat and pig) in
human(20) , all of which are on the light subunit. However, the
human mutant
-glutamyl transpeptidase in which these corresponding
residues were replaced by Ala was found to be almost fully active, and
the inactivation by acivicin was as rapidly for the mutant as for the
wild-type enzyme; thus, these residues are not essential for
catalysis(20) . These findings suggested that another hydroxyl
group reacts with acivicin initially, followed by transfer of acivicin
moiety to the aforementioned residues. Such a primarily reactive
hydroxyl group is likely to be identical to the serine residue involved
in formation of
-glutamyl enzyme intermediate because of
structural similarity between the
-glutamyl moiety and acivicin,
and because of the requirement of a nucleophile for both the
-glutamyl substrate and acivicin to react with the enzyme.
In
the current study, we selected the conserved serine residues as
candidates for mutagenesis by using amino acid sequence alignment of
the light subunits of human, rat, pig and two bacterial -glutamyl
transpeptidases. These candidates were examined by site-specific amino
acid substitutions in order to identify the hydroxyl group(s)
responsible for inactivation by acivicin and for catalysis.
Figure 1:
Sequence alignment of the light
subunits of -glutamyl transpeptidases from a variety of species.
Shaded boxes show the amino acid residues conserved among all species.
The residue number for the human enzyme is given above the aligned
sequences. The numbers on the right correspond to the residue number of
each species. Arrow heads indicate the serine residues examined in the
human enzyme.
The K values of Ser-451 and -452 mutant
-glutamyl transpeptidases for the donor substrate were only about
4 and 2-times higher than that of the wild-type enzyme, respectively.
However, their V
values were 40 times lower (Table 2). Furthermore, their double mutation resulted in
significant decrease of V
value to about 0.005%
of that of the wild type, but the K
for the donor
increased only modestly. Similarly, large decreases in V
values, without significant alteration of K
, were also observed in hydrolysis of L-
-glutamyl-p-nitroanilide by these
serine-substituted mutants (Table 2).
The decreased activity
in the Ser-451 and -452 mutants is not likely to be due to an inability
to use glycylglycine as an acceptor substrate because the presence of
20 mM glycylglycine substantially enhanced the V values of both of these mutants (Table 2). Thus, the decrease in activity (transpeptidation and
hydrolysis) appears to be due to hindrance in chemical step involving
the donor substrate, L-
-glutamyl-p-nitroanilide.
Since the substrate K
values are not significantly
increased in the mutants, the amino acid substitutions at Ser-451 and
-452 do not seem to affect steady-state binding of the substrates,
which suggests that these mutations do not cause a gross conformational
alteration. These results show that both Ser-451 and -452 are important
in catalysis.
The reaction rates in transpeptidation of Ser-451,
-452 and their double mutants showed saturation by the addition of only
0.5 mM of the acceptor. Hence, substitutions at Ser-451 -452
and Ser-451/452 have decreased K values for the
acceptor. Their slower rates of formation of the enzyme species
(
-glutamyl enzyme) necessary for the binding of the second
substrate (glycylglycine) would cause a relatively low concentration of
the
-glutamyl enzyme species, leading to lower K
values for the second substrate. These considerations are
consistent with impairment of step from the enzyme-first substrate
complex to the
-glutamyl enzyme in a ping-pong mechanism proposed
for the enzyme (10, 36) .
Figure 2:
Schematic representation of the active
site of human -glutamyl transpeptidase. A, formation of
-glutamyl enzyme; B, Covalent adduct of acivicin to the active
site; C, Inhibition by L-serine as a competitive inhibitor; D,
Inhibition by serine-borate complex as an inhibitory transition-state
analog.
The inhibition constants of the wild-type
and mutant enzymes by L-serine or serine-borate complex are
given in Table 5. All enzymes examined were inhibited in a
competitive manner. The mutant enzymes in which Ser-385, 413 or 425
were replaced by Ala exhibited the same K values
for L-serine and for serine-borate complex as for the
wild-type enzyme. In the presence of saturating borate (10
mM), the K
value of the wild type for L-serine decreased to 0.4% of that of L-serine alone.
This decrease in K
is attributed to the formation
of a reversible linkage between the borate portion of the serine-borate
complex and the hydroxyl group(s) of the enzyme. As expected from their K
values for L-
-glutamyl-p-nitroanilide in the kinetic study, K
values of Ser-451 and -452 mutants for L-serine in the absence of borate were only 5.5 and 3.8 fold
higher than the wild type, respectively.
Nevertheless, the usual
decrease in K by addition of borate to the
Ser-451, -452 and the double mutants was not as large as for the
wild-type enzyme. Thus, substitution at Ser-451 and at Ser-452 greatly
abolished (about 300 and 8 fold, respectively) inhibition by the
serine-borate complex. Even at the concentration of L-serine
higher than that of the borate, these inhibitions depended on the
serine concentration. The borate appears to react with the complex of
the enzyme and the serine because the borate needs the hydroxyl groups
disposed appropriately to form the linkages. Therefore, Ser-451 and
-452 residues appear to be necessary to form the linkage between the
enzyme and borate. Moreover, inhibition of the double mutant appears to
depend only on the serine portion of the serine-borate complex because
the K
values of the double mutant for
serine-borate complex is much greater (>1,000 fold) than that of the
wild type, while the K
value of the double mutant
for L-serine (without borate) is similar to the K
value of the mutant for the serine-borate
complex. This is in contrast to the wild-type and other active mutant
enzymes. Additionally, the K
of the double mutant
for L-serine alone was relatively higher than expected from
its K
values for the
-glutamyl substrate,
compared with the other mutants. This may be due to the lack of a
hydrogen bond between the hydroxyl groups of L-serine as an
inhibitor and the two serine residues, Ser-451 and -452, of the enzyme.
These results suggest that both Ser-451 and -452 are accessible to the
-carbonyl carbon of the
-glutamyl substrate.
The studies reported here indicate that Ser-451 or -452, or
both, are essential for catalysis of -glutamyl transpeptidase, and
that neither of these participate in the binding of the
-glutamyl
substrate. The results also suggest that these serine residues are
positioned in the active site to allow interaction with the
-carbonyl carbon of the
-glutamyl moiety of the substrate. In
addition to their spatial arrangement, the serine residues are
nucleophilic so as to give rise to the nucleophilic substitution at the
imide moiety of acivicin in the covalent-adduct formation. It also
suggests that these residues would be primarily responsible for
interaction with acivicin prior to the transfer to Ser-406 as proposed
in previous studies(20) . This seems analogous to the
intramolecular transfer of the acyl group as found in fatty acid
synthase(42) . These findings are consistent with the
suggestion that both Ser-451 and -452 are catalytic nucleophiles
involved in formation of
-glutamyl enzyme intermediate. However,
it is also possible that these residues may stabilize the transition
state in a manner similar to that of the oxyanion hole found in serine
proteases(37) .
The rate of hydrolysis of L--glutamyl-p-nitroanilide even by the double
mutant, although virtually inactive, is still 2,000 times faster than
without enzyme; the k
of the double mutant was
1.7
10
s
, while
non-enzymatic hydrolysis was 9.5
10
s
. This enhancement of the reaction rate may
be achieved by a reaction pathway that is different from the normal
mechanism. In such a pathway, a catalytic nucleophile may not be
necessary. This possibility is similar to that for a subtilisin (a
serine protease) mutant, where the catalytic serine was replaced by
alanine. The mutant enzyme facilitated the hydrolysis of the
acyl-p-nitroanilide substrate 3,000 times faster than in the
absence of enzyme(38) . This alternative mechanism of
subtilisin for hydrolysis of the substrate without the catalytic serine
is based on a detailed structure of the active site.
The motif,
Pro-Leu-Ser-Ser
-Met, is one of the most
conservative regions in the complete sequences of
-glutamyl
transpeptidase from a variety of species. Further, the Ser-452 residue
in the human enzyme is the only serine residue conserved among all
-glutamyl transpeptidases for which the primary sequences are
known and the two related enzymes, human
-glutamyl
transpeptidase-related enzyme (39) and Pseudomonas cephalosporin acylase(40) . The later two are distinct
from
-glutamyl transpeptidase but have significant sequence
homology. These comparisons suggest that these serine residues are of
critical importance to enzyme function. Our data obtained using
site-directed mutagenesis are in accord with this consideration in
terms of sequence homology. Replacement of Ser-452 by threonine
resulted in almost the same catalytic properties as substitution by
alanine (data not shown). These results show that the hydroxyl residue
at position 452 is restricted to only a serine for enzyme function.
In general, the common sequence motif, Gly-Xaa-Ser-Xaa-Gly (Ala), is
conserved at the region including the catalytic serine among
serine-class hydrolases(41) . Nevertheless, this motif was not
found after alignment of the whole sequences of a family of
-glutamyl transpeptidases. If the serine residues identified here
actually act as catalytic nucleophiles in the mechanism of
-glutamyl transpeptidase, the enzyme may utilize either
nucleophile to attack the carbonyl moiety of the substrate, and might
be distinct from ordinary serine-class hydrolases.
Our current
studies identify two serine residues required for catalysis, Ser-451
and -452 in human -glutamyl transpeptidase. These are tentatively
proposed as catalytic nucleophiles or alternatively the stabilizing
residues of the transition state (Fig. 3). Further structural
studies are still needed to ascertain the functions of these serine
residues in catalysis. Our findings, however, provide valuable
information on the active site chemistry of
-glutamyl
transpeptidase and its related enzymes.
Figure 3:
Possible roles of Ser-451 and -452 in the
catalytic mechanism of human -glutamyl transpeptidase. A, as
catalytic nucleophiles; B, stabilization of the transition state.
Arg-107 and Asp-423 are also shown, as proposed in previous studies to
bind the
-glutamyl moiety (ref. 13 and 19). X indicates the
leaving group of the substrate.