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INTRODUCTION |
Glutamate dehydrogenase
(GDH1; EC 1.4.1.3) is a
family of enzymes that catalyze the reversible deamination of
L-glutamate to 2-oxoglutarate using NAD+,
NADP+, or both as coenzyme (1). Since the pathology of the
disorders associated with GDH defects is restricted to the brain, the
enzyme may be of particular importance in the biology of the nervous system. The importance of the pathophysiological nature of the GDH-deficient neurological disorders has attracted considerable interest (2). The enzyme isolated from one of the patients with a
variant form of multisystem atrophy displayed a marked reduction of one
of the GDH isoproteins (3). Although the origin of the GDH polymorphism
is not known, it has been reported that the presence of differently
sized mRNAs and multiple gene copies for GDH occur in the human
brain (4). A novel cDNA encoded by an X chromosome-linked
intronless gene also has been isolated from human retina (5). However,
it is not known whether the distinct properties of the GDH isoproteins
are essential for the regulation of glutamate metabolism. Therefore, it
is essential to have a detailed structural and functional description
of the various types of brain GDH to elucidate the pathophysiological nature of the GDH-deficient neurological disorders.
Mammalian GDH is composed of six identical subunits, and the regulation
of GDH is very complex (1). It has been a major goal to identify the
substrate and regulatory binding sites of GDH. It is the only recent
years that the three-dimensional structure of GDH from microorganisms
is available (6, 7). Recently, crystallization of bovine liver GDH was
reported for the first time from the mammalian sources (8). However,
remarkably little is known about the detailed structure of mammalian
GDH, especially the brain enzymes. Although regulatory and substrate
binding sites of GDH have been reported, the results are quite
controversial. Several classical chemical probes have been used to
attempt resolution of these binding sites. The studies using classical
chemical probes to identify the NADH and GTP binding site within bovine
liver GDH, however, gave a wide scatter of modified residues throughout most of the proposed three-dimensional structure of GDH. For instance, the NADH binding site was proposed to be modified by an ATP analogue at
Cys319 (9), by a GMP probe at Met169 and
Tyr262 (10), and by the adenosine analogue at
Lys420 and Tyr190 (11). It seems, therefore,
that the base moiety has not been effective at directing the site of
modification by classical chemical probes.
Alternatively, identifying nucleotide binding sites of variety of
proteins has been advanced by the use of nucleotide photoaffinity analogues that selectively insert into a site upon photoactivation with
ultraviolet light. For instance,
[32P]2N3NAD+ was shown to be a
valid active-site probe for several proteins (12-15). The ATP binding
site of adenylate kinase and creatine kinase and the protein unique to
cerebrospinal fluids of Alzheimer's patients successfully have been
identified using 2N3ATP and 8N3ATP (16). We
previously isolated and characterized two soluble forms of glutamate
dehydrogenase isoproteins (designated GDH I and GDH II) from bovine
brain (17-20) and identified the GTP and NAD+ binding site
of the GDH isoproteins using [32P]8N3GTP and
[32P]2N3NAD+, respectively (21,
22). Very recently, Stanley et al. (23) have reported that
the hyperinsulinism-hyperammonemia syndrome is caused by mutations in
GDH gene that affects enzyme sensitivity to GTP-induced inhibition. The
mutations identified in the patients with hyperinsulinism and
hyperammonemia (23) exactly lie within a sequence of 15 amino acids
that we previously suggested to contain GTP binding site of the brain
GDH isoproteins (21). On the other hand, the location of the mutations
on GDH in those patients is quite distinct from the GTP binding site
identified by using the classical chemical probe (24). These results
prove selectivity and specificity of the photoaffinity probe as a valid
active-site probe.
In the present work, we report the identification of an ADP binding
site in the overall sequence by a combination of peptide analysis and
photolabeling with [
-32P]8N3ADP. The
results obtained using photoaffinity probe place the ADP binding domain
within a proposed catalytic cleft defined in the crystal structure
(25).
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EXPERIMENTAL PROCEDURES |
Materials--
NADP+, NAD+, NADH,
2-oxoglutarate, ADP, GTP, L-glutamate, aluminum chloride,
and L-1-tosylamido-2-phenylethyl chloromethyl
ketone-treated trypsin were purchased from Sigma. 8N3ADP
was purchased from RPI Corp. [
-32P]8N3ADP
was synthesized by the method as described previously (26, 27).
Epoxy-activated iminodiacetate-Sepharose 6B was obtained from Sigma.
All other chemicals and solvents were reagent grade or better.
Enzyme Purification and Assay--
The GDH isoproteins were
purified from bovine brains by the method developed in our laboratory
(17) and were homogeneous as judged by Coomassie-stained gradient
SDS-polyacrylamide gel electrophoresis. Only homogeneously purified GDH
isoproteins were used unless otherwise indicated. GDH activity was
measured spectrophotometrically in the direction of reductive amination
of 2-oxoglutarate by following the decrease in absorbance at 340 nm as
described previously (17). Activation of GDH I and GDH II by ADP and
8N3ADP were examined by incubating the enzymes with ADP or
8N3ADP at various concentrations in 50 mM
triethanolamine, pH 8.0, at 25 °C. At intervals after the initiation
of the activation, aliquots were withdrawn for the assay of enzyme activity.
Photolabeling of GDH Isoproteins--
Photolabeling of GDH
isoproteins were performed by the method of Shoemaker and Haley (28)
with a slight modification. For saturation studies, GDH I and GDH II
(0.1 mg each) in 10 mM Tris acetate, pH 8.0, were
separately incubated with various concentrations of
[
-32P]8N3ADP in Eppendorf tubes for 5 min
with a hand-held UV lamp at a distance of 4 cm. For competition
studies, 0.1 mg of GDH isoproteins were incubated with various
concentrations of ADP for 10 min in the same buffer prior to the
addition of 100 µM [
-32P]8N3ADP and then allowed to incubate
with the photoprobe for 5 min as described above. The samples were then
irradiated with a hand-held 254-nm UV lamp for 90 s twice at
4 °C. The reaction was quenched by the addition of ice-cold
trichloroacetic acid (final 7%). The reaction mixtures were kept on
ice bath for 30 min and then centrifuged at 10,000 × g
for 15 min at 4 °C. The pellets were washed and resuspended with 10 mM Tris acetate, pH 8.0. The remaining free photoprobe, if
any, was further removed from the protein by exhaustive washing using
Centrifree (Amicon), and 32P incorporation into protein was
determined by liquid scintillation counting.
Tryptic Digestion of Photolabeled GDH Isoproteins--
To
determine the site modified by
[
-32P]8N3ADP, 2.0-mg samples of each GDH
isoprotein in 10 mM Tris acetate, pH 8.0, were separately incubated with 100 µM
[
-32P]8N3ADP for 5 min at 4 °C.
Glutamate (5 mM), NAD+ (1.4 mM),
NADP+ (0.1 mM), and GTP (0.1 mM)
were also included in the incubation mixture to saturate their
respective binding sites. The mixtures were irradiated for 90 s
twice. In some experiments, GDH I and GDH II were photolyzed in the
presence of additional 1 mM ADP to validate that the
isolated peptide(s) was specific for the ADP binding site and could be
protected from photomodification. The reaction was quenched by the
addition of ice-cold trichloroacetic acid (final 7%) and kept at
4 °C for 30 min. The protein was precipitated by centrifugation at
10,000 × g for 15 min at 4 °C, and the pellet was
resuspended in 75 mM NH4HCO3, pH
8.5, containing 2 M urea. GDH isoproteins were proteolyzed
by the addition of 15 µg of trypsin and kept at room temperature for
3 h, after which 15 µg of trypsin was added again. After 3 more
hours at room temperature, 20 µg of trypsin was added, and the
digestion mixture was kept at 25 °C overnight.
Isolation of the Photolabeled Peptide and Protein
Sequencing--
An immobilized aluminum chromatography procedure (29)
modified to isolate photolabeled peptides was used as described
elsewhere (16, 28). For preparation of the immobilized aluminum column, 1.5 ml of an epoxy-activated iminodiacetate-Sepharose 6B was chelated to Al3+ by slow passage through 20 ml of 50 mM
AlCl3. The resin was then washed successively with
H2O and 50 mM ammonium acetate, pH 5.9 (buffer
A); 0.5 M NaCl in buffer A (buffer B); buffer A, 2 M urea in buffer A (buffer C); buffer A, 5 mM
glutamate in buffer A (buffer D); and buffer A. Buffer A was added to a
fraction of the tryptic digestion mixture (1:1), and dithiothreitol was
added to a final concentration of 1 mM. The pH was adjusted
to 6.0, and the tryptic digested peptides were applied to the column.
The column was washed with approximately 15 ml of buffers A, B, A, C,
A, D, and A, respectively (0.5 mM/min). The photolabeled
peptides were eluted with 5 mM KH2PO4 in 50 mM ammonium acetate,
pH 8.0. The absorbance of the fractions was measured at 220 nm and the
photoincorporation was determined by liquid scintillation counting.
Fractions from immobilized aluminum column were further purified by
reversed-phase HPLC. Fractions containing photolabeled peptides were
desalted, freeze-dried, resuspended in 0.1% trifluoroacetic acid, and
subjected to reversed-phase HPLC using an Waters C18 column
on the same HPLC system. The mobile system consisted of 0.1%
trifluoroacetic acid solution and 0.1% trifluoroacetic acid, 80%
acetonitrile solvent system. The gradient for HPLC was 0-10 min, 0%
acetonitrile; 10-60 min, 0-80% acetonitrile; 60-70 min, 80%
acetonitrile at a flow rate 0.5 ml/min. HPLC fractions containing photolabeled peptides were pyridylethylated by the method described elsewhere (30) and sequenced by the Edman degradation method as
described previously (17).
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RESULTS |
Activation of GDH Isoproteins by ADP and
8N3ADP--
The vertebrates GDHs have been well known to
be regulated by ADP. To show that 8N3ADP could mimic the
regulatory properties of ADP, the photoanalogue should be able to
activate GDH as well as ADP. As Fig. 1
shows, both ADP and 8N3ADP activated the activities of the
GDH isoproteins, although maximal activity with 8N3ADP was
about 75% of maximal ADP-stimulated activity. These results were
consistent with an earlier report showing interaction of 8-azido-ADP
with bovine liver glutamate dehydrogenase (31). These results show that
the azidonucleotide, 8N3ADP, is able to elicit the similar
biological effects on GDH isoproteins as the natural nucleotide,
ADP.

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Fig. 1.
Activation of GDH isoproteins by ADP and
8N3ADP. The effects of ADP and 8N3ADP on
GDH I and GDH II were examined in the direction of reductive amination
of 2-oxoglutarate with NADH as a coenzyme at pH 8.0. , GDH I + ADP;
, GDH I + 8N3ADP; , GDH II + ADP; , GDH II + 8N3ADP.
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Saturation and Competition of Photoinsertion--
To show
specificity of the photoprobe-protein interaction, saturation of
photoinsertion should be observed. To demonstrate saturation effects
with the photoprobe, the enzymes were photolabeled with increasing
concentrations of [
-32P]8N3ADP in the
presence of glutarate. Under the experimental conditions described,
saturation of photoinsertion with
[
-32P]8N3ADP occurred at around 80~100
µM photoprobe with apparent Kd values
of 25 µM and 40 µM for GDH I and GDH II,
respectively (Fig. 2). The apparent
Kd values were also measured from the
double-reciprocal plot (data not shown), and the results were almost
identical from those obtained in Fig. 2. In all photolabeling experiments, the ionic strength was kept low to enhance binding affinity, as we have observed in general that the lower the ionic strength the tighter the binding of nucleotide photoaffinity probes and
the more efficient the photoinsertion. Therefore, the reported apparent
Kd values obtained from photoaffinity labeling should be interpreted considering that photolabeling is done under conditions that enhance binding site occupancy. Under saturating conditions, 0.95 mol and 0.90 mol of photolabel/mol of GDH subunit were
introduced for GDH I and GDH II, respectively. The results in Fig. 2
indicate the saturability of ADP specific site of GDH isoproteins with
this photoprobe and therefore decrease the possibility of nonspecific
photoinsertion.

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Fig. 2.
Saturation of
[ -32P]8N3ADP
phosphoincorporation into GDH isoproteins. GDH I and GDH II in the
reaction buffer were photolyzed with the indicated concentrations of
[ -32P]8N3ADP, and 32P
incorporation into protein was determined by liquid scintillation
counting (see "Experimental Procedures" for details). Relative
32P incorporations were expressed relative to each control.
100% relative 32P incorporation corresponded to 75,000 and
71,000 cpm for GDH I and GDH II, respectively. , GDH I; , GDH
II.
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To further demonstrate specific labeling of GDH isoproteins, the
enzymes were photolabeled with
[
-32P]8N3ADP in the presence of increasing
ADP concentrations. As shown in the results of the competition
experiments (Fig. 3), increasing ADP
concentration decreased the photolabeling of 100 µM
[
-32P]8N3ADP. The apparent
Kd values of this interaction were 33 µM and 55 µM for GDH I and GDH II,
respectively. NAD+ was able to inhibit photoinsertion, but
not as effectively as ADP (Table I). ATP
and GTP had only a minor effect on the photoinsertion (Table I). When
concentrations of the nucleotides were reduced, these effects were less
extensive. These results show the specificity of
[
-32P]8N3ADP and the utility of this probe
as a good candidate for determining the ADP binding site.

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Fig. 3.
The effect of ADP on
[ -32P]8N3ADP
phosphoincorporation into GDH isoproteins. GDH I and GDH II in the
reaction buffer were photolyzed with 100 µM
[ -32P]8N3ADP in the presence of the
indicated concentrations of ADP. Relative 32P incorporation
into protein was determined and expressed as described in the legend to
Fig. 2. , GDH I; , GDH II.
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Table I
Effects of nucleotides on photolabeling of brain GDH isoproteins with
[ -32P]8N3ADP
GDH isoproteins were incubated with the indicated concentrations of
various nucleotides for 1 min in 50 mM triethanolamine, pH
8.0, at 25 °C prior to the addition of 100 µM
[ 32P]8N3ADP. Relative 32P incorporation
into protein was determined as described in the legend to Fig. 3.
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Tryptic Digestion of Photolabeled Proteins and Isolation of the
Photolabeled Peptide--
To identify the peptides modified by
[
-32P]8N3ADP, GDH isoproteins were
photolabeled twice in the absence and presence of 1 mM ADP
and digested with trypsin. To reduce any possible nonspecific labeling
and at the same time to optimize the specific labeling of the enzymes,
100 µM [
-32P]8N3ADP was
used, which is the concentration at which photoinsertion approaches
saturation. In addition, 5 mM glutamate, 0.1 mM
NADP+, 1.4 mM NAD+, and 0.1 mM GTP were included in the reaction mixture to reduce the
nonspecific binding of [
-32P]8N3ADP to
those sites, although they decreased the level of photoinsertion of
[
-32P]8N3ADP (Table I). The photolabeled
GDH isoproteins were separated from most of the noncovalently bound
nucleotide by acid precipitation and proteolyzed by trypsin. The
digested samples were applied to an immobilized aluminum column. The
immobilized aluminum column retained over 90% of the radioactivity
(Fig. 4), and the photolabeled peptide
was eluted with 5 mM KH2PO4, pH
8.0. One major radioactive peak was recovered from the immobilized
aluminum column. Over 85% of the loaded radioactivity from the sample
was associated with the eluates from the immobilized aluminum column
(fractions 20-30 in Fig. 4). ADP was able to reduce
[
-32P]8N3ADP photoinsertion into this
peak. When 1 mM ADP was originally present in the
incubation mixture, more than 90% of the radioactivity of the peak was
eliminated as shown in Fig. 4, and the UV absorption peak eluted in
fractions 20-26 was dramatically decreased (omitted in Fig. 4 for
clarity purposes). This result indicates that the radioactive peak
represent a peptide in the ADP binding domain of the GDH
isoproteins.

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Fig. 4.
Purification of tryptic peptides from GDH I
photolabeled with
[ -32P]8N3ADP on an
aluminum chelate column. GDH I was photolabeled with
[ -32P]8N3ADP as described under
"Experimental Procedures" in the absence and presence of 1 mM ADP, and the mixture was subjected to an immobilized
aluminum chromatography. Each 1-ml fraction was monitored for
absorbance at 220 nm by UV spectrophotometer and for radioactivity by
liquid scintillation. The plain solid line represents the
absorbance at 220 nm of the sample photolabeled in the absence of 1 mM ADP. The plot also represents the radioactivity profiles
from the aluminum chelate column of the sample photolabeled in absence
( ) and the presence ( ) of 1 mM ADP. Fractions 1-5
and 6-20 represent the fractions collected while loading and washing,
respectively. Fractions 20-30 represent fractions eluted with 5 mM K2HPO4.
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When the flow-through fractions from aluminum column were subjected to
separation on a reversed-phase HPLC column, multiple peaks were
observed as determined with UV-absorption spectrophotometer at 220 nm,
indicating that many of the peptides were not retained on the resin
(data not shown). When the radioactive eluates from the immobilized
aluminum column were subjected to reversed-phase HPLC, one major
radioactive peak and one minor nonradioactive peak were clearly
observed (Fig. 5). Although some
radioactivity was found in the HPLC flow-through and wash fractions,
over 90% of the total radioactivity coeluted with the major peak. The
radioactivity associated with the HPLC flow-through fractions
represents unbound probe including any decomposition products of
photoadduct produced as peptide binds to the HPLC column matrix. These
flow-through fractions were subjected to analysis and no significant
amounts of amino acids were detected. The radioactive fractions were
collected and identified by sequence analysis. GDH II gave almost
identical chromatographic profiles to GDH I on both immobilized
aluminum chromatography and reversed-phase HPLC, even though the
intensities of the radioactivity was slightly lower than those of GDH I
(data not shown). These results demonstrates that the
microenvironmental structures of the two GDH isoproteins are very
similar to each other. The photolabeled peptides of GDH II were,
therefore, treated and sequenced by the same method as described
above.

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Fig. 5.
Reversed-phase HPLC purification of tryptic
peptides eluting from an aluminum chelate column. The radioactive
eluates from the aluminum chelate column were loaded onto a
C18 reversed-phase column, eluted with an acetonitrile
gradient (dashed line) at a flow rate of 0.5 ml/min, and
monitored at 220 nm (plain solid line). One-minute fractions
were collected. The solid line with closed
circles represents radioactivity. Levels of 32P were
determined by liquid scintillation counting in aqueous phase.
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Sequence Analysis and Amino Acid Composition of the Photolabeled
Peptide--
As shown in Table II, the
amino acid sequence analysis revealed that the photolabeled peptide of
the GDH isoproteins contained the amino acid sequence,
EMSWIADTYASTIGHYD and EMSWIADTYASTIGHYDIN for GDH I and GDH II,
respectively. The sequences obtained were also compared with those of
various GDHs (Table III). Since these are
tryptic digests, it was expected to produce a sequence ending with Arg
or Lys. The amino acid composition of the photolabeled peptide revealed
that the peptide had a composition that was compatible with that of the
tryptic peptide spanning residue Glu179-Lys205
of the amino acid sequence of the mature human GDHs (Table
IV). Photolabeling of the peptide was
prevented over 90% by the presence of 1 mM ADP during
photolysis. These results demonstrate selectivity of the photoprobe for
the ADP binding site and suggest that the peptide identified using the
photoprobe is located in the ADP binding domain of the brain GDH
isoproteins.
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Table II
Amino acid sequence of the [ 32P]8N3ADP-modified
peptide
The amino acids are denoted by the single-letter code.
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DISCUSSION |
In the present work, we identified an adenine binding domain
peptide of the ADP binding site of two GDH isoproteins from bovine brain using photoaffinity probe
[
-32P]8N3ADP and peptide analysis. The
specificity of 8N3ADP and the utility of this probe as a
good candidate for determining the ADP site were demonstrated by the
following. First, in the absence of activating light,
8N3ADP was able to activate GDH similarly to ADP. The
ability to mimic a native compound before photolysis has an advantage
over determination of the enzyme function after modification. A
previous study showed that 8N3ADP and ADP induced similar
effects on the structure of GDH (31). This is consistent with the
results in Fig. 1. Second, the data showing decreased photoinsertion by
addition of ADP demonstrates that photoinsertion occurs only by the
bound form of [
-32P]8N3ADP. Also,
reduction by other nucleotides was not as effective as that observed
with ADP. This indicates that proximity controls photoinsertion and
that the residues modified are within the adenine binding domain.
Third, saturation of photoinsertion at concentrations corresponding to
that expected from the reversible binding affinities also strongly
supports the site being labeled is within the binding domain. Finally,
their selectivity and specificity of 8N3ADP have been
successfully utilized to locate the specific base binding domains of
nucleotide binding site peptides of many proteins (12-15, 21, 22).
To identify the site of photoinsertion, the photolabeled GDH
isoproteins were digested with trypsin and the peptides were purified
by aluminum chelate chromatography. The immobilized aluminum chromatography was able to successfully retain the radiolabel of
photolabeled peptides and greatly reduces the possibility of any
nonphotolabeled peptide coeluting on HPLC with the photolabeled peptide, which could give misleading results. Also, excess protease might be added without a problem with this technique since we have
observed no detectable peptide fragments of the protease being
retained. Once photolabeled peptides are bound to the metal chelate
resin, they can be washed with 0.1 M ammonium acetate solutions containing 0.5 M NaCl or 2 M urea to
elute any peptides that may be bound to the resin other than through
phosphate-metal interactions. The flow-through fractions from aluminum
chelate affinity column had a multiple of peptides (data not shown),
while the phosphate eluate contained only two UV peaks from HPLC, one of which was radioactive (Fig. 5). The use of metal chelate
chromatography enhances the recovery of photolabeled peptides, because
it reduces the number of separation steps. These results indicate that
aluminum chelate chromatography could prove to be an effective way of
isolating azidonucleotide-photomodified peptides.
The crystal structure of Clostridium symbiosum GDH has been
aligned with the primary sequence of the bovine liver GDH (25). The
structures of C. symbiosum and mammalian GDHs were suggested to be similar due to considerable identity and the conservation of 13 glycine residues, which probably conserves the structure among species,
and to consist of two domains. The first domain has been proposed to
contain the catalytically important residues, while the second domain
contains a
-
-
motif that is responsible for coenzyme binding
(32, 33). The primary sequence of bovine liver GDH also suggests the
presence of a
-
-
motif corresponding to the motif within the
bacterial subunit. Previously, we identified GTP binding site of bovine
brain GDH isoproteins as the peptide containing residues
Ile440-Arg459 using
[
-32P]8N3GTP, an effective biomimic of GTP
in inhibiting GDH activity (21). Since only the vertebrate GDHs contain
the sequence corresponding to Ile440-Arg459,
and only GDHs from these sources are regulated by GTP, it seems likely
that the regulatory GTP binding domains are located in this region. Our
results were clearly consistent with those reported by Shoemaker and
Haley (14), who identified the GTP binding site of bovine liver GDH by
photoaffinity labeling. Very recently, Stanley et al. (23)
have reported that the hyperinsulinism-hyperammonemia syndrome is
caused by mutations in GDH gene that affects enzyme sensitivity to
GTP-induced inhibition. The sensitivity of GDH to inhibition by GTP was
a quarter of the normal level in the patients with sporadic
hyperinsulinism-hyperammonemia syndrome. The mutations identified in
the patients with hyperinsulinism and hyperammonemia (23) exactly lie
within a sequence of 19 amino acids
(Ile440-Arg459) that we previously suggested
to contain GTP binding site of the brain GDH isoproteins (21). The
mutations in the patients with sporadic cases impaired enzyme
sensitivity to GTP-induced inhibition but did not affect basal or
ADP-stimulated enzyme activity (23). The ADP and GTP binding sites have
been thought to be distinct but proximal and possibly overlapping (34).
Both nucleotides also elicit different conformational changes within
the enzyme (35).
The similarities between the structures of NAD+ and ADP
could contribute to the difficulties in identifying the specific ADP binding site. In the present work, the photoprobe
[
-32P]8N3ADP is shown to mimic ADP
kinetically (Fig. 1) and specifically identify the ADP binding site of
GDH I and GDH II (Table II). The results also indicate that there are
high levels of sequence identity between GDHs compared and the
photolabeled peptides corresponds to the tryptic peptide spanning
residue Glu179-Lys205 of the amino acid
sequence of the mature human GDHs (Table III and Table IV). The peptide
within this region were specifically protected from photoinsertion by
competing amounts of ADP. Comparisons of this sequence with the
bacterial enzyme suggest that these peptides lie within the first
globular domain. It was reported that the affinity of ADP and
8N3ADP to GDH is not as tight as that of NAD+,
GTP, and their corresponding photoaffinity probes (28). This is most
likely due to fewer points of protein-nucleotide contact, which would
account for loose binding. While certain residues of the photolabeled
peptides showed decreases compared with other residues, the exact
residues photolabeled within the adenine ring domain within the ADP
binding site remain uncertain. Our results are clearly consistent with
those reported by Shoemaker and Haley (28), who identified the ADP
binding site of bovine liver GDH using
[
-32P]8N3ADP and
[
-32P]2N3ADP. Data obtained using
photoaffinity probes (13, 14, 21, 22) place all of the base binding
domains of NAD+, GTP, and ADP at different locations within
a proposed catalytic cleft defined in the structure by Teller et
al. (25).
In contrast to our approach, several classical chemical probes have
been used to attempt resolution of the binding sites and have shown
quite discrepant results. For instance, the ADP binding site was
proposed to be modified by an two different AMP analogues at
His82 (36) and Arg459 (37). These two residues
are outside the catalytic cleft. Similar results with discrepancy using
classical chemical probes were also reported by the same research group
to identify other regulatory sites within bovine liver GDH. The NADH
binding site was proposed to be modified by an ATP analogue at
Cys319 (9), by a GMP probe at Met169 and
Tyr262 (10), and by the adenosine analogue at
Lys420 and Tyr190 (11). The GTP binding site
was also proposed to be modified by
5'-p-(fluorosulfonyl)benzoyl-1,N6-ethenoadenosine
at Tyr262 (24). It is not clear why a hydrophobic
adenosine-containing probe
5'-p-(fluorosulfonyl)benzoyl-1,N6-ethenoadenosine
preferentially bind and react at a hydrophilic GTP binding site and not
react at the other adenosine binding sites. Above all, the GTP binding
site identified by the classical chemical probes are quite distinct
from the GTP binding site (Ile440-Arg459)
observed in the patients with hyperinsulinism and hyperammonemia (23).
It seems, therefore, that the base moiety has not been effective at
directing the site of modification by classical chemical probes.
Chemically reactive probes, due to their long-lived reactive state,
have an increased opportunity to react with the most nucleophilic residues within an enzyme and may not necessarily react with a less
reactive or nonreactive residue that may be located within the binding
domain. This is especially likely if they display low affinity for the
binding site being studies. Their lack of specificity may be the reason
for the wide three-dimensional distribution of the residues identified
using classical chemical probes as being in the NADH inhibitory site of
GDH (9, 10). An analysis of the three-dimensional structure of the
mammalian enzyme should supplement the understanding of the nature and
location of these regulatory sites.
Partial deficiency of GDH isoproteins has been reported in some
patients with cerebellar degeneration, suggesting that the enzymes are
important in brain function (3, 4). The existence of the
hyperinsulinism-hyperammonemia syndrome also highlights the importance
of GDH in the regulation of insulin secretion (23, 38) and indicates
that GDH has an important role in regulating hepatic ureagenesis (39).
Further studies of GDH and its isotypes may provide explanations for
the absence of central nervous system symptoms due to hyperammonemia
(40). As many proteins have functions distinct from those for which
they were originally identified, the other roles of GDH isoproteins
have been reported. For instance, a membrane-bound form of GDH
possesses a microtubule binding activity (41), and GDH reacts as a
RNA-binding protein and shows a possible role in regulation of
transcription (42, 43). Recently, Cavallaro et al. (44) have
identified GDH as one of the late memory-related genes in the
hippocampus. It would appear that we have just begun to unravel the
mystery of GDH and their role in biological system. To our knowledge,
comparison of the detailed structure of active sites and regulatory
sites of any GDH isoproteins rarely has been reported. In conclusion,
the work presented here clearly identifies the ADP binding site of the
brain GDH isoproteins in the overall sequence.