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Originally published In Press as doi:10.1074/jbc.M007908200 on September 25, 2000
J. Biol. Chem., Vol. 275, Issue 51, 40365-40370, December 22, 2000
Molecular Modeling and Site-directed Mutagenesis Define the
Catalytic Motif in Human  -Glutamyl Hydrolase*
Karen J.
Chave,
Ivan E.
Auger,
John
Galivan, and
Thomas J.
Ryan
From the Division of Molecular Medicine, Wadsworth Center, New York
State Department of of Health, Albany, New York 12201-0509
Received for publication, August 29, 2000, and in revised form, September 20, 2000
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ABSTRACT |
Human  -glutamyl hydrolase (hGH) is a central
enzyme in folyl and antifolylpoly--glutamate metabolism, which
functions by catalyzing the cleavage of the -glutamyl chain of
substrates. We previously reported that Cys-110 is essential for
activity. Using the sequence of hGH as a query, alignment searches of
protein data bases were made using the SSearch and TPROBE programs.
Significant similarity was found between hGH and the glutamine
amidotransferase type I domain of Escherichia coli
carbamoyl phosphate synthetase. The resulting hypothesis is that the
catalytic fold of hGH is similar to the folding of this domain in
carbamoyl phosphate synthetase. This model predicts that Cys-110 of hGH
is the active site nucleophile and forms a catalytic triad with
residues His-220 and Glu-222. The hGH mutants C110A, H220A, and E222A
were prepared. Consistent with the model, mutants C110A and H220A were
inactive. However, the Vmax of the E222A hGH
mutant was reduced only 6-fold relative to the wild-type enzyme. The
model also predicted that His-171 in hGH may be involved in substrate
binding. The H171N hGH mutant was found to have a 250-fold reduced
Vmax. These studies to determine the catalytic
mechanism begin to define the three dimensional interactions of hGH
with poly--glutamate substrates.
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INTRODUCTION |
-Glutamyl hydrolase
(GH)1 (EC 3.4.19.9), which
hydrolyses the -glutamyl conjugates of folic acid and antifolates,
is a key enzyme in the metabolism of folic acid and the pharmacology of
antifolates. Folate is required as a cofactor by several enzymes in the
de novo biosynthesis of DNA precursors and several amino acids. Antifolates (for example, methotrexate (MTX)) have been the
traditional treatment for many cancers. When the cell takes up folates
or antifolates, they are poly--glutamylated by the enzyme
folylpolyglutamate synthetase (EC 6.3.2.17) (1). These polyglutamates
are retained intracellularly and are generally better substrates or
inhibitors of the target enzymes (2-4). GH alters these properties by
catalyzing the removal of the polyglutamate chain (1, 5, 6).
Although GH is known to cleave folylpolyglutamates, ultimately yielding
folylmonoglutamate and glutamate, its role in the intracellular
folate-dependent pathways remains unclear. Similarly, the
role of GH in folate deficiencies is ambiguous. Increased GH activity
(7, 8) or decreased folylpolyglutamate synthetase activity (9-12) can
produce resistance to MTX in vitro. Recently, the ratio of
folylpolyglutamate synthetase and GH activities has been demonstrated
to be an indicator of MTX polyglutamylation in acute lymphocytic
leukemia (13), and inherent resistance to MTX in acute myelogenous
leukemia (14) may be due to an increased GH activity. However,
clarification of the role of GH in these outcomes is unavailable.
Therefore, definition of the active site residues and mechanism of
human GH (hGH) will be necessary steps in developing specific
inhibitors to assess its cellular function.
Despite the important role of GH in folate function and antifolate
therapeutics, no structural studies of the enzyme have been carried
out. The catalytic mechanism of GH is not known. It has been
established that the rat and human GH proteins are inhibited by
iodoacetic acid (1, 15), suggesting that at least one cysteine is
important for activity. In a previous study (16) using site-directed
mutagenesis, we altered the cDNA for hGH to encode four different
proteins, each with one of four cysteine residues changed to alanine.
Three of the mutant proteins had activities similar to wild-type hGH
and were inhibited by iodoacetic acid, whereas the C110A mutant protein
had no activity. Cys-110 is conserved among the human, rat, and mouse
GH amino acid sequences. These results indicate that Cys-110 is
essential for enzyme activity and suggest that GH is a cysteine
peptidase. The present study extends this earlier work by using
comparative molecular modeling to predict the catalytic fold and
presents the first molecular model for hGH. The catalytic fold of hGH
is predicted to be similar to that of the glutamine amidotransferase
type I (GATase) domain of the small subunit of carbamoyl-phosphate
synthetase (CPS, EC 6.3.5.5) from Escherichia coli (eCPS).
In the model, Cys-110 is proposed as the active site nucleophile, and
His-220 and Glu-222 are predicted to be the other amino acids in the
putative catalytic triad. The model also predicts that His-171 is
involved in substrate binding but not catalysis. Site-directed
mutagenesis of these conserved amino acids followed by characterization
of the purified mutant proteins was used to test the predictions of the
model. Determination of the active site fold is the first step in
defining the structure of hGH in order to develop specific inhibitors
that can be used to understand the role of this enzyme in folate
homeostasis and antifolate therapy.
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EXPERIMENTAL PROCEDURES |
Generation of the Model for the Catalytic Fold of Human
-Glutamyl Hydrolase--
A two-step process was used to generate
the sequence alignment model. First, using the sequence of hGH as the
query, a search of Swiss-Prot plus TrEMBL data bases was made using the
program SSearch (17), which uses the Smith-Waterman algorithm to
identify statistically significant sequence similarities. The set of
protein sequences obtained from this search was then used as a starting set for analysis using the program TPROBE (18). TPROBE performs a Gibbs
alignment of this set, and it generates motif models based on this
alignment. These alignment motifs are next used to search the
non-redundant data bases to extract additional sequences that fit the
alignment model. TPROBE then purges sequences that are closely related
so that each pair of sequences in the set has a BLAST maximal segment
pair score less than a cutoff score. For our analysis, the cutoff score
was set at 150. TPROBE then performs a Gibbs alignment on this set and
generates a new alignment model. This process is repeated until the
number of recruited sequences does not increase significantly. As
described unde "Results," this process identified seven motifs of
the GATase family of proteins. The motifs of the GATase domains of
proteins of known structure were then aligned with the corresponding
motifs in hGH. The SEGMOD module of GeneMine (Molecular Applications
Group, Palo Alto, CA) was then used to generate a molecular model by
homology between hGH and the most similar sequence with known
structure. This procedure uses a data base search strategy and energy
minimization algorithm (19, 20). The coordinates of the known structure
are used as a basis to derive predicted coordinates of the target
protein. The coordinates of the parent are combined with coordinates of target homologous segments from the structural data base to construct 10 initial models. Then an average model based on these 10 is derived.
Energy minimization is employed to reduce steric overlap and produce
the final predicted structure.
Expression and Purification of Wild-type and Mutant hGH
Proteins--
The cDNA for the mature forms of wild-type and C110A
hGH were subcloned from pET24a (16) into the NdeI and
BamHI sites of the pET28b vector (Novagen, Madison, WI)
using standard molecular biology protocols (21). This added an
N-terminal His tag to the proteins. The final expressed protein had the
N-terminal sequence Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-Met before Arg-1 of the previously described sequence of the mature enzyme
(15). Site-directed mutagenesis was performed with the QuickChange kit
(Stratagene, La Jolla, CA) according to the manufacturer's protocol
using the expression vector for the wild-type enzyme as the template,
except that 100 ng of each oligonucleotide was used, and the annealing
temperature was reduced to 53 °C. Oligonucleotides used for
mutagenesis of wild-type hGH to H220A, E222A, and H171N were as
follows: GHH220A+, 5'-TGT CCA GTG GGC TCC AGA GAA AGC-3'; GHH220A , 5'-GCT TTC TCT GGA GCC CAC TGG ACA-3'; GHE222A+,
5'-GTG GCA TCC AGC GAA AGC ACC TT-3'; GHE222A, 5'-AAG GTG
CTT TCG CTG GAT GCC AC-3'; GHH171N+, 5'-CTG CCA ATT
TCA ATA AGT GGA GCC TCT CCG-3'; GHH171N- 5'-CGG AGA GGC TCC
ACT TAT TGA AAT TGG CAG-3'. The nucleotides altered during
site-directed mutagenesis are underlined.
After mutagenesis, plasmids were purified using the QIAfilter plasmid
midi kit (Qiagen, Valencia, CA) following the manufacturer's protocol,
and the entire hGH open reading frame was sequenced on an ABI 377 sequencer (PerkinElmer Life Sciences).
Wild-type enzyme or mutants of hGH were expressed in an E. coli expression system (Novagen) as described previously (16). Wild-type or mutant hGH proteins were purified to homogeneity by a
two-step procedure. The clarified sonicate from 1 liter of culture was
purified by nickel chelate chromatography on a HisBind column (6.3 × 1.0 cm) at either room temperature or 4 °C following the
manufacturer's protocol (Novagen). Eluate containing hGH protein was
dialyzed into 0.05 M sodium acetate buffer, pH 5.5, containing 0.05 M 2-mercaptoethanol, 1 M NaCl,
and 1 mM EDTA. Aliquots (2 ml) were further purified by gel
filtration chromatography at 4 °C on a Sephacryl S-200 column
(2.5 × 95 cm) (Amersham Pharmacia Biotech) in the same buffer
with a flow rate of 20 ml/h. Fractions (2.5 ml) containing hGH protein
were pooled, and the protein was concentrated using an Amicon
(Beverley, MA) stirred cell with a YM-10 membrane. Protein
concentrations were determined as described previously (16).
Enzyme Assays--
Enzyme activity was measured using the
substrate 4-NH2-10-CH3PteGlu2
(MTXG2, Shirck Laboratories, Jona, Switzerland) as
described previously (16), and kinetic constants were determined as
described previously (16). The results presented are an average of at least two protein preparations and three sets of assays per protein preparation.
Secondary Structure Determination by Circular Dichroism--
To
determine the secondary structure of wild-type and mutant hGH proteins,
measurements were made on a Jasco 720 spectropolarimeter (Japan
Spectroscopic Ltd, Tokyo, Japan). Three scans were made at 20 nm/min
between 260 and 200 nm in a 0.05-, 0.02-, or 0.01-cm cell at 25 °C.
After subtraction of the buffer spectra, the data were converted to
molar ellipicity units. The Selcon program (22) was used to calculate
the  helix,  sheet, turns, and random structure.
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RESULTS |
Construction of the Model--
By using the SSearch and TPROBE
programs, a statistically significant similarity was found between the
GATase type I group of proteins and hGH. TPROBE generated an alignment
model with seven motifs. A total of 391 sequences were identified as
members of this superfamily. The Sequence Logo alignment model (23) illustrated in Fig. 1 uses 47 of these
sequences since TPROBE purges sequences that are too closely related.
The numbering used corresponds to that for mature hGH (15). In
particular, the well characterized GATase catalytic residues cysteine,
histidine, and a putative third residue, glutamate, are strongly
conserved in the sequence alignment model. Among the set of sequences
that belong to the identified superfamily, the small subunit of eCPS (24), GMP synthetase from E. coli (25), and anthranilate
synthase from Sulfolobus solfataricus (26) have known high
resolution x-ray structures with Protein Data Bank accession numbers
1jdb, 1gpm, and 1qdl, respectively. A molecular model of hGH is presented in Fig. 2. The image was
generated with the program MolScript (27) and rendered by the program
Raster3D (28). The model was generated by first aligning amino acid
sequences in hGH with the corresponding sequence in eCPS and then using eCPS as a template structure for the SEGMOD module of GeneMine. The
other structures were aligned as well, but the modeling software selected eCPS, which is the most similar sequence to hGH, with a 17%
similarity. The superimposition of the -carbon atoms of the hGH
model with 1jdb chain C, 1qdl chain B, and 1gpm chain A had root mean
square deviation C values of 1.02, 4.76, and 8.37 angstroms,
respectively. The regions that can be predicted with confidence are
those that in the alignment model have a maximum a
posteriori probability (MAP) score that is positive or close to
zero maximum. In hGH, these regions are residues 95-124, 199-223, 59-82, 21-48, and 157-181, corresponding to 254-283, 332-356, 226-249, 190-217, and 298-322 in 1jdb chain C. Each individual motif
is colored in Fig. 2 in order of descending statistical significance as
follows: orange 95-124, blue 199-223,
gold 59-82, salmon 21-48, sea green
157-181. Red regions represent those sequences outside of
the five significant motifs. There are four regions in hGH that are
sequence insertions with respect to eCPS. The sequence insertion in hGH
of residues 142-159 has corresponding but slightly smaller insertion
regions in 1gpm chain A and 1qdl chain B compared with 1jdb chain C. The other three insertions are unique to hGH, since they do not occur
in the other structures. These insertions are shown in red
in Fig. 2.
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Fig. 1.
Sequence logo of the motifs identified by
TPROBE. The numbering used is that of mature hGH. The motifs in
order of statistical significance are hGH residues 95-124, 199-223,
59-82, 21-48, 157-181, 244-253, and 142-148. The logo was
constructed from an alignment of 47 sequences. The vertical
axis represents the amount of information (in bits) that this
position holds. The height of the one-letter residue symbol is
proportional to the information bit of the residue at that position.
The sequence logo was generated using WebLogo (23).
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Fig. 2.
Molecular model of human
-glutamyl hydrolase. The model was generated
using the TPROBE alignment and the SEGMOD module in GeneMine. Red
regions are those outside the five significant motifs identified
by SEGMOD. The significant motifs are in color: residues 95-124
(orange), residues 199-223 (blue), residues
59-82 (gold), residues 21-48 (salmon), and
residues 157-181 (sea green). The residues corresponding to
the proposed catalytic triad of hGH are Cys-110 (yellow),
His-220 (pale blue), and Glu-222 (bisque).
Catalytically important His-171 in hGH is shown in pale
blue. A, the entire molecular model of hGH.
B, magnification of the area containing the catalytic amino
acids.
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As a test of the proposed structure, a model structure for the subunit
of GMP synthetase, 1gpm chain A, was calculated using the same
procedure. The percent similarity of the alignment between 1gpm chain A
and 1jdb chain C is 19%. Since this value is close to the 17%
similarity between hGH and 1jdb chain C, the model structure for 1gpm
chain A can be used as a positive control to validate the molecular
modeling of hGH. The region 7-207 of 1gpm chain A was modeled using
1jdb chain C as a template structure. The region starts with the first
motif identified by TPROBE and ends with the last motif. The region
includes the two non-significant motifs. A structural superimposition
of the five significant motifs between the 1gpm chain A model and the
known 1gpm chain A structure (25) had a root mean square deviation C
of 3.86 Å, and the overall superimposition had a root mean square
deviation C of 4.99 Å. The overall fold of the model was similar to
the known structure. As expected, the model of the loop, 1gpm chain A
117-131, corresponding to the insertion in 1gpm, is not accurate, and
the five significant motifs (1gpm chain A 71-100, 160-184, 44-67,
9-36, and 129-153) overlap quite well.
Predictions of the Model--
The model predicts that Cys-110 in
hGH (yellow in Fig. 2) is analogous to Cys-269 in eCPS. eCPS
contains two domains and catalyzes the synthesis of carbamoyl phosphate
from bicarbonate, glutamine, and two molecules of MgATP. The smaller
subunit is the site of glutamine hydrolysis to produce ammonia for
delivery to the larger subunit. eCPS and other members of the GATase I
family of enzymes contain a Cys-His-Glu catalytic triad (25). Cys-269
in eCPS functions as the active site nucleophile in the small subunit, attacking the -carbonyl of glutamine to form a glutamyl thioester intermediate (29). His-220 (pale blue in Fig. 2) and Glu-222 in hGH (bisque in Fig. 2) are predicted to be the other two
amino acids in the proposed catalytic triad corresponding to His-353 and Glu-355 in eCPS. The alignment model also predicts that His-171 in
hGH (pale blue in Fig. 2) is analogous to His-312 in eCPS.
Site-directed Mutagenesis of hGH--
The addition of an
N-terminal HisTag to hGH enabled the purification to homogeneity of
large quantities of wild-type and mutant proteins from the E. coli expression system (Fig. 3). The
wild-type enzyme and mutant proteins reacted with a polyclonal antibody raised against hGH expressed in insect cells (Fig. 3) (15). Analysis of the secondary structures of wild-type and mutant hGH proteins by circular dichroism (Table
I) indicated that they were
essentially the same, demonstrating that the site-directed mutagenesis
did not significantly alter the protein folding.
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Fig. 3.
SDS-12.5% polyacrylamide gel electrophoresis
analysis of purified wild-type and mutant hGH proteins.
A, gel (2.0 µg/lane) stained with 0.25%
Coomassie Blue. B, Western blot analysis (0.2 µg/lane) using a polyclonal antibody (1:10,000 dilution)
against hGH expressed in insect cells (15). 1, wild-type
enzyme; 2, C110A mutant; 3, H220A mutant;
4, E222A mutant; 5, H171N mutant.
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Table I
Secondary structure prediction of wild-type and mutant hGH proteins
Circular dichroism spectra were generated between 260 and 200 nm. The
data were converted to molar ellipicity units and used in the program
Selcon (22) to determine the percentage of -helix, -sheet, turn,
and random structure. Results are the mean ± S.D. of 3-6
spectra.
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Enzyme Kinetics of Wild-type and Mutant hGH Proteins--
The
purified wild-type and mutant hGH proteins were assayed for activity
with MTXG2 (Table II). The
kinetic constants obtained for the wild-type hGH protein with an
N-terminal His tag were similar to previous results with a non-His-tag
hGH protein (16), suggesting that the addition of the N-terminal His
tag had no effect on hGH activity. As predicted by the molecular
modeling and previous results in a non-His-tag expression system (16), mutation of Cys-110 to alanine in the His-tag construct produced inactive enzyme. Mutation of His-220 to alanine also produced an
inactive enzyme, confirming the critical role of this amino acid in
catalysis. Mutation of Glu-222 to alanine produced active enzyme, with
a Km similar to that of the wild-type hGH and a
6-fold reduced Vmax (Table II). Mutating His-171
to asparagine produced an enzyme with a 250-fold reduction in
Vmax (Table II) but an unchanged
Km.
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Table II
Kinetic constants for wild-type and mutant hGH proteins
Km and Vmax were determined using
variable concentrations of 4-NH2-10-CH3PteGlu2
as substrate and capillary electrophoresis to determine product (15).
The results presented are the mean and S.D. of at least two protein
preparations and three assays per preparation.
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DISCUSSION |
The proposed molecular model predicted the catalytic triad based
on homology with the GATase domain of eCPS. In agreement with our
previous study of the four hGH cysteine residues in which Cys-110 was
identified as essential for activity (16), expression of the C110A hGH
mutant in an E. coli expression system using a construct
with a HisTag attached to the N terminus produced inactive enzyme.
Amino acid Cys-110 was confirmed as necessary for catalytic activity
and predicted by homology to Cys-269 in eCPS to be the active site
nucleophile that attacks the -carbonyl of the susceptible Glu-Glu
bond in poly--glutamate substrates to form a thioester intermediate.
A similar mutation in eCPS produced protein that bound but did not
hydrolyze glutamine (30).
The production of an inactive hGH mutant by mutating His-220 to alanine
is consistent with the predicted role in the catalytic mechanism. The
equivalent residue in eCPS (His-353) is suggested to have several
roles: to activate the cysteine nucleophile, to protonate the leaving
group, and to activate the water molecule that attacks the thioester
intermediate (29, 31-34). Mutating His-353 to asparagine in eCPS
produced an enzyme with greatly reduced activity, which still bound
glutamine (31). The same mutation in a mammalian CPS yielded a protein
with a 164-fold lower kcat, a 4-fold higher
Km, and an unmeasurably low rate of
thioester formation (32). The mutation of the equivalent histidine in
the GATase domain of anthranilate synthase decreased both activity and
sensitivity to 6-diazo-5-oxo-L-norleucine, suggesting that
this histidine residue plays a role in the formation of the thioester
intermediate (35).
The results presented here indicate that Glu-222 is not catalytically
essential in hGH. When this residue was mutated to alanine there was a
6-fold reduction in Vmax, indicating that the
E222A hGH mutant retains more activity than the equivalent mutant of eCPS. The thermal stability for the hGH E222A mutant protein was also
decreased, and a lower yield of purified E222A was observed (results
not shown). These observations are consistent with results obtained by
site-directed mutagenesis of the putative third member of the catalytic
triad of papain (36). Mutation of this asparagine to glutamine and
alanine reduced the kcat/Km
3.4- and 150-fold, respectively. It was also found that these mutants
had increased aggregation, proteolytic susceptibility, and decreased thermal stability, suggesting that this residue was also structurally important.
The role of Glu-355 as a member of a catalytic triad in eCPS has
recently been the subject of some debate. Glu-355 is conserved in all
known GATase type I sequences and was proposed to be a member of the
catalytic triad based on analogy to other hydrolyzing enzymes. The
location of this conserved glutamate within hydrogen-bonding distance
of His-353 in the crystal structures of eCPS (24) and the equivalent
histidine in E. coli GMP synthetase (25) added strength to
this hypothesis. Only recently has site-directed mutagenesis been
performed to test this hypothesis. When this residue was mutated to
glycine in mammalian CPS, an enzyme was generated that had an unaltered
Km but 47-fold lower kcat
(32) and poor thioester formation. An E355A mutation in eCPS (33)
produced an enzyme with a 47-fold higher Km in
glutamine-dependent reactions but an unaltered
Vmax. In p-aminobenzoate synthetase (37), a 35-fold increased Km, 4-fold decreased
Vmax, and reduced thioester formation were
observed after mutation of the equivalent glutamate residue. The
retention of some activity when the active site glutamate was mutated
to alanine argues against Glu-355 being absolutely essential for
catalysis, but poor thioester formation suggests that it may have an
indirect role in catalysis. The absolute conservation of this glutamate
residue in GATase domains and also in GH proteins suggests a critical
role for this residue. In hGH, Glu-222 may play more of a role in
structural integrity than in catalysis.
The model predicted that His-171 in hGH would function like His-312 in
eCPS. His-312 in eCPS has been suggested to be involved in substrate
binding but not catalysis (31). Mutating His-171 in hGH to asparagine
reduced enzyme activity. Asparagine was chosen as it is
sterically conservative and may maintain some hydrogen-bonding patterns but cannot perform the acid/base reactions characteristic of
the the imidazole ring. However, unlike the equivalent mutation in
eCPS, Vmax and not Km was
altered in hGH. This absence of a reduction in Km
suggests that His-171 in hGH functions differently than His-312 in
eCPS. The large reduction in Vmax for the H171N
mutation implies some unknown role in the catalytic mechanism of hGH.
The crystal structure of eCPS indicates that the imidazole ring of
His-312 points away from the catalytic triad and is approximately 5 Å from the catalytic cysteine (24). His-312 was proposed to be involved
in glutamine binding rather than catalysis based on biochemical
analysis of an H312N mutant eCPS protein. This mutant had a 47-fold
increase in the Km for the glutaminase reaction but
an unaltered Vmax (31). However, in the crystal
structure of the H353N eCPS mutant, where the thioester intermediate
has been trapped (29), there are no apparent electrostatic interactions
between His-312 and the substrate. Therefore, the role of this residue
in substrate binding remains unknown.
The hypothesis that the two enzymes use a similar catalytic mechanism
is consistent with the similarity of the biochemical reactions
catalyzed by hGH and the GATase domain of eCPS. It has previously been
shown that the products of the hGH-catalyzed reaction on
folypolyglutamates are glutamate or di--glutamate (15). The amide
group hydrolyzed in the polyglutamate chain is the N-terminal of one
glutamate linked to the -carbonyl of the preceding glutamate. The
GATase domain of eCPS removes the -NH2 from glutamine to produce glutamate and ammonia (24-26, 29-34). The reaction proceeds by attack of the active site cysteine on the -carbonyl of glutamine to form a thioester intermediate and ammonia (24, 29, 33, 34). It can
be envisioned that if the catalytic mechanism of eCPS is applied to
hGH, the active site cysteine would attack the -carbonyl of the
poly--glutamate to form the thioester, and the -linked glutamate
would be released. hGH has been shown to be inhibited by glutamine
analogues such as azaserine and 6-diazo-5-oxo-L-norleucine (38), also suggesting that the active site could be similar to the
glutamine binding GATase domain of eCPS.
These results indicate that hGH is a member of the cysteine peptidase
family of enzymes with Cys-110 as the active site nucleophile and
His-220 as the proton donor. The catalytic fold appears to be similar
to that of the GATase type I domain, with His-171 having an essential
but as yet undetermined role in enzyme activity.
Ollis et al. (39) identify the / hydrolase fold common
to several hydrolytic enzymes with different catalytic function and
phylogenetic function. The x-ray structure of GMP synthetase (25)
indicates that the GATase type I domain has a fold different than that
of the / hydrolase fold. However, some of the elements of the
/ hydrolase fold are retained. For example, the "nucleophile elbow" is present. In this structural feature, the catalytic cysteine is the last amino acid of a -sheet and the first of an -helix, forcing it into an unusual orientation (39). To maintain this structure, the amino acids +2 and 2 around the catalytic cysteine must be small (39). In hGH residues 108 and 112 are both glycines. Therefore, hGH may also contain a nucleophile elbow. X-ray
crystallography studies are under way to test this hypothesis and to
determine the role of His-171. The results presented here define the
active site fold and catalytic residues of hGH and should facilitate the design of high affinity inhibitors specific for GH to test whether
inhibition of GH activity would lead to increased retention of folyl-
and antifolylpolyglutamates within the cell.
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ACKNOWLEDGEMENTS |
We thank the Molecular Genetics Core of the
Wadsworth Center for oligonucleotide synthesis and DNA sequencing, the
Biochemistry Core for the use of the circular dichroism instrument, and
the Computational Molecular Biology and Statistics Core for
computational support.
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FOOTNOTES |
*
This work was supported by NCI, National Institutes of
Health Grants CA25933 (to J. G.) and CA82425 (to T. J. R.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 518-474-6193;
Fax: 518-473-2900; E-mail: thomas.ryan@wadsworth.org.
Published, JBC Papers in Press, September 25, 2000, DOI 10.1074/jbc.M007908200
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ABBREVIATIONS |
The abbreviations used are:
GH, -glutamyl
hydrolase;
hGH human GH, CPS, carbamoyl-phosphate synthetase;
eCPS
E. coli CPS, GATase, glutamine amidotransferase;
MTX, methotrexate (4-NH2-10-CH3PteGlu);
MTXG2, MTX diglutamate
(4-NH2-10-CH3PteGlu2);
Pte, pteroyl.
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, pp. 135-190, John Wiley & Sons, Inc., New York
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Galivan, J.
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Schirch, V.,
and Strong, V. W.
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