Identification of an NAD+ Binding Site of Brain Glutamate Dehydrogenase Isoproteins by Photoaffinity Labeling*

Photoaffinity labeling with [32P]nicotinamide 2-azidoadenosine dinucleotide (2N3NAD+) was used to identify the NAD+ binding site within two types of glutamate dehydrogenase isoproteins (GDH I and GDH II) isolated from bovine brain. In the absence of photolysis, 2N3NAD+ is a substrate for the GDH isoproteins. When the enzymes were covalently modified by photolysis in the presence of saturating amounts of photoprobe, about 50% inhibition of the GDH activities was observed. Photoinsertion of probe was increased by GTP or glutarate and decreased by NAD+ or ADP. With the combination of immobilized boronate affinity chromatography and reversed-phase HPLC, photolabel-containing peptides generated with trypsin were isolated. This identified a portion of the adenine ring binding domain of GDH isoproteins as the region containing the sequence, CIAVGXSDGSIWNPDGIDPK for both GDH isoproteins, corresponding to Cys270 through Lys289 of the amino acid sequence of well known bovine liver GDH. The Xindicates a position for which no phenylthiohydantoin-derivative could be assigned. The missing residue, however, can be designated as a photolabeled glutamate since the sequences including the glutamate residue in question have a complete identity with those of the other GDH species known. Photolabeling of these peptides was prevented by the presence of NAD+ during photolysis. These results demonstrate selectivity of the photoprobe for the NAD+binding site and suggest that the peptide identified using the photoprobe is located in the NAD+ binding domain of the brain GDH isoproteins. Both amino acid sequencing and compositional analysis identified Glu275 as the site of photoinsertion.

Glutamate is a major excitatory neurotransmitter (1) and is known to be involved in the pathogenesis of human degenerative disorders because of its neurotoxic potentials (2,3). One enzyme central to the metabolism of glutamate is glutamate dehydrogenase (GDH 1 ; EC 1.4.1.3), which catalyzes the reversible deamination of L-glutamate to 2-oxoglutarate using NAD ϩ or NADP ϩ . Mammalian GDH is composed of six identical subunits, and the regulation of GDH is very complex (4). It has been a major goal to identify the substrate and regulatory binding sites of GDH. It is only in recent years that the three-dimensional structure of GDH from microorganisms is available (5,6). Very recently, crystallization of bovine liver GDH was reported for the first time from the mammalian sources (7). However, remarkably little is known about the detailed structure of mammalian GDH, especially the brain enzymes.
Even though there are several reports suggesting the regulatory or substrate binding site, 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 sites within bovine liver GDH, however, gave a wide scatter of modified residues throughout most of the proposed threedimensional structure of GDH. For instance, the NADH binding site was proposed to be modified by an ATP analogue at Cys 319 (8), by a GMP probe at Met 169 and Tyr 262 (9), and by the adenosine analogue at Lys 420 and Tyr 190 (10). 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 a 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, [ 32 P]2N 3 NAD ϩ was shown to be a valid active site probe for several proteins (11)(12)(13)(14). The ATP binding site of adenylate kinase and creatine kinase and the protein unique to cerebrospinal fluids of Alzheimer's patients have been identified successfully using 2N 3 ATP and 8N 3 ATP (15,16). The ADP regulatory site and the GTP binding site of bovine liver GDH also have been identified using [ 32 P]2N 3 NAD ϩ and [ 32 P]8N 3 GTP, respectively (17,18).
Because the pathology of the disorders associated with GDH defects is restricted to the brain, this enzyme may be of particular importance in the biology of the nervous system. Hussain et al. (19) detected four different forms of GDH isoproteins from human cerebellum of normal subjects and patients with neurodegenerative disorders. The enzyme isolated from one patient with a variant form of multisystem atrophy displayed marked reduction of one of the GDH isoproteins (19). The isoproteins are differentially distributed in the two catalytically active isoforms of the enzyme (20). The origin of the GDH polymorphism is not known. Current studies showed the presence of four different sized mRNA and multiple gene copies for GDH in the human (20,21). A novel cDNA encoded by an X chromosome-linked intronless gene was also isolated from human retina (22). Although the existence of brain GDH isoproteins has been recognized, the comparative studies of the GDH isoproteins from any sources are far less encompassing in protein function and structure. Further characterization of the structure and function of these various types of brain GDH is needed to elucidate the pathophysiological nature of the GDHdeficient neurological disorders.
We previously have isolated two soluble forms of glutamate dehydrogenase isoproteins (designated GDH I and GDH II) from bovine brain (23) and identified the GTP binding site of the GDH isoproteins using [ 32 P]8N 3 GTP (24). We also have reported the regulatory properties of the bovine brain GDH I and GDH II (25)(26)(27). In the present work, we report the identification of an NAD ϩ binding site in the overall sequence by a combination of peptide analysis and photolabeling with [ 32 P]2N 3 NAD ϩ . The results obtained using photoaffinity probe place the NAD ϩ binding domain within a proposed catalytic cleft defined in the crystal structure (28).
Enzyme Purification and Assay-The GDH isoproteins were purified from bovine brains by the method developed in our laboratory (23) 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 (23).
Photolabeling of GDH Isoproteins-Photolabeling of GDH isoproteins was performed by the method of Kim and Haley (12) with a slight modification. For saturation studies, GDH I and GDH II (100 g each) in 10 mM Tris acetate, pH 8.0, containing 12 mM glutarate were incubated separately with various concentrations of [ 32 P]2N 3 NAD ϩ in Eppendorf tubes for 5 min. For competition studies, 100 g of GDH isoproteins were incubated with various concentrations of NAD ϩ for 10 min in the same buffer prior to the addition of 50 M [ 32 P]2N 3 NAD ϩ and then allowed to incubate with the photoprobe for 5 min as described above. The samples were 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 in an ice bath for 30 min and centrifuged at 10,000 ϫ 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 removed from the protein by exhaustive washing using Centrifree (Amicon), and 32 P incorporation into protein was determined by liquid scintillation counting.
Tryptic Digestion of Photolabeled GDH Isoproteins-To determine the site modified by [ 32 P]2N 3 NAD ϩ , 2.0-mg samples of each GDH isoprotein in 10 mM Tris acetate, pH 8.0, were incubated separately with 50 M [ 32 P]2N 3 NAD ϩ in the presence of 1 mM GTP, 12 mM glutarate, and 10 M EDTA for 5 min at 4°C. The mixtures were irradiated for 90 s twice. 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 ϫ g for 15 min at 4°C, and the pellet was resuspended in 75 mM NH 4 HCO 3 , pH 8.5, containing 2 M urea. GDH isoproteins were proteolyzed by the addition of 15 g of trypsin and kept at room temperature for 3 h after which 15 g of trypsin was added again. After 3 more hours at room temperature, 20 g of trypsin was added, and the digestion mixture was kept at 25°C overnight. To validate that the isolated peptide(s) was specific for the NAD ϩ binding site and could be protected from photomodification, GDH I and GDH II were photolyzed in the presence of 150 M NAD ϩ and proteolyzed as described above.
Isolation of the Photolabeled Peptide and Protein Sequencing-The tryptic digested peptides were applied to a boronate column equilibrated with 0.1 M ammonium acetate, pH 9.0. Unmodified peptides were washed with the same buffer, and photolabeled peptides were eluted with a 200-ml gradient of pH 9.0 -5.0 in 0.1 M ammonium acetate. The absorbance of the fractions was measured at 220 nm, and the photoincorporation was determined by liquid scintillation counting. The fractions containing photolabeled peptides were desalted, freezedried, resuspended in 0.1% trifluoroacetic acid, and subjected to reversed-phase HPLC using a Waters C 18 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 (29) and sequenced by the Edman degradation method as described previously (23). 3 NAD ϩ on GDH Activity-In the absence of photolysis, 2N 3 NAD ϩ is a substrate for the GDH isoproteins ( Table I). The ability of 2N 3 NAD ϩ to inhibit the activities of GDH isoproteins by photoinsertion on irradiation under different conditions was investigated. GDH activities were not affected by UV light under these conditions. When the enzymes were covalently modified by photolysis in the presence of saturating amounts of 2N 3 NAD ϩ , about 50% inhibition of the GDH activities was observed (Table I). When the enzymes were photolyzed in the presence of 500 M NAD ϩ in addition to 50 M 2N 3 NAD ϩ , about 88ϳ90% of the initial activities of GDH isoproteins remained. These results suggest that 2N 3 NAD ϩ was photoinserting into a NAD ϩ binding site on GDH isoproteins in a specific manner.

Effects of 2N
Active site involvement was also supported by the effects of GTP and ADP on photoinsertion (Table II). It is well known that GTP increases the binding of NAD ϩ while ADP weakens the binding with or without glutarate present (30,31). Similar regulatory effects of these nucleotides were observed using [ 32 P]2N 3 NAD ϩ when the enzymes were photolabeled with the photoprobe in the presence of GTP or ADP (Table II). Compared with a control, the addition of 1 mM GTP caused an approximate 2.3-fold increase of photoinsertion whereas the presence of 1 mM ADP caused an approximate 55% reduction. This reduction by ADP was lower than an 80% decrease in photoinsertion caused by the addition of NAD ϩ . When concentrations of the nucleotides were reduced, these effects were less extensive.
Saturation and Competition of Photoinsertion-To show specificity of the photoprobe-protein interaction, saturation of photoinsertion should be observed. The binding of NAD ϩ to GDH has been studied by equilibrium dialysis and initial rates (32). In the presence of glutarate, NAD ϩ was bound more tightly (31). To demonstrate saturation effects with the photoprobe, the enzymes were photolabeled with increasing concentrations of [ 32 P]2N 3 NAD ϩ in the presence of glutarate. Under the experimental conditions described, saturation of photoinsertion with [ 32 P]2N 3 NAD ϩ occurred at around 40 M photoprobe with apparent K d values near 10ϳ15 M for GDH isoproteins (Fig. 1). In all photolabeling experiments, the ionic  3 NAD ϩ GDH isoproteins were photolabeled in 10 mM Tris acetate, pH 8.0, containing 10 M EDTA, 12 mM glutarate, and 50 M 2N 3 NAD ϩ . An aliquot was taken and assayed as described under "Experimental Procedures." The presence or absence of light prior to assay is indicated by a (ϩ) or (Ϫ), respectively.

Additions hv
Remaining activity a GDH I GDH II 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, when interpreting the reported apparent K d values obtained from photoaffinity labeling one should consider that photolabeling is done under conditions that enhance binding site occupancy. The results in Fig. 1 indicate the saturability of the NAD ϩ -specific site of GDH isoproteins with this photoprobe and therefore decrease the possibility of nonspecific photoinsertion.
To further demonstrate specific labeling of GDH isoproteins, the enzymes were photolabeled with [ 32 P]2N 3 NAD ϩ in the presence of increasing NAD ϩ concentrations. As shown in the results of the competition experiments (Fig. 2), NAD ϩ was able to protect photolabeling from 10 M [ 32 P]2N 3 NAD ϩ at concentrations in the range of known K d values (32). Approximately, 80% protection was observed with 150 M NAD ϩ for both GDH isoproteins (Fig. 2). These results show the specificity of [ 32 P]2N 3 NAD ϩ and the utility of this probe as a good candidate for determining the NAD ϩ binding site.
Tryptic Digestion of Photolabeled Proteins and Isolation of the Photolabeled Peptide-To identify the peptides modified by [ 32 P]2N 3 NAD ϩ , GDH isoproteins were photolabeled twice in the absence and presence of 150 M NAD ϩ and digested with trypsin. To reduce any possible nonspecific labeling and at the same time to optimize the specific labeling of the enzymes, 50 M [ 32 P]2N 3 NAD ϩ was used, which is the concentration at which photoinsertion approaches saturation. In addition, 1 mM GTP and 12 mM glutarate were included in the reaction mixture since they were shown to increase the binding affinity of NAD ϩ (30, 31) and [ 32 P]2N 3 NAD ϩ (Table II) to GDH isopro-teins. 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 a boronate column equilibrated with 0.1 M ammonium acetate, pH 9.0. All of the unlabeled peptides were eluted with ammonium acetate buffer, pH 9.0, in the void volume, whereas photolabeled peptides were selectively retained on the column. The radioactive peptides were eluted with a pH gradient of 9.0 -5.0 (Fig. 3). One major radioactive peak (indicated by the arrow in Fig. 3) around pH 6.5 was recovered from the column. NAD ϩ was able to reduce [ 32 P]2N 3 NAD ϩ photoinsertion into this peak. When 150 M NAD ϩ was originally present in the incubation mixture, more than 90% of the radioactivity of the peak was eliminated as shown in Fig. 3. This result indicates that the radioactive peak represents a peptide in the NAD ϩ binding domain of the GDH isoproteins.
When the radioactive eluates from the boronate column were subjected to reversed-phase HPLC, one major radioactive peak (fractions 25-27) was clearly observed (Fig. 4). Although some radioactivity was found in the HPLC flow-through and wash fractions, over 90% of the total radioactivity co-eluted with the major peak. The radioactivity associated with the HPLC flowthrough 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 peptides (fractions [25][26][27] were collected and identified by sequence analysis. GDH II gave almost identical chromatographic profiles to GDH I on both boronate and reversed-phase HPLC column, even though the intensities of the radioactivity were slightly higher than those of GDH I (data not shown). These results demonstrate 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.
Sequence Analysis of the Photolabeled Peptide of GDH I and GDH II-The amino acid sequence analysis revealed that the peak fractions contained the amino acid sequence CIAVGXS-DGSIWNPDGIDPK for both GDH isoproteins. The sequences obtained were also compared with those of various GDHs (Table III). As judged by comparison with the well known amino acid sequence of bovine liver GDH, this site was identified as residues 270 -289 of bovine liver GDH. The X indicates a position for which no phenylthiohydantoin-derivative could be assigned. The missing residue, however, can be designated as a photolabeled glutamate since the sequences including the glutamate residue in question have a complete identity with those FIG. 1. Saturation of [ 32 P]2N 3 NAD ؉ phosphoincorporation into GDH isoproteins. GDH I and GDH II in the reaction buffer were photolyzed with the indicated concentrations of [ 32 P]2N 3 NAD ϩ , and 32 P incorporation into protein was determined by liquid scintillation counting (see "Experimental Procedures" for details). Relative 32 P incorporations were expressed relative to each control. q, GDH I; Ⅺ, GDH II.  2. The effect of NAD ؉ on [ 32 P]2N 3 NAD ؉ phosphoincorporation into GDH isoproteins. GDH I and GDH II in the reaction buffer were photolyzed with 50 M [ 32 P]2N 3 NAD ϩ in the presence of the indicated concentrations of NAD ϩ . Relative 32 P incorporation into protein was determined and expressed as described in Fig. 1. q,GDH I; Ⅺ, GDH II. of the other GDH species known. 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 residues 270 -289 with the exception that there was a significant reduction in Glu (data not shown). Photolabeling of the peptide was prevented by the presence of NAD ϩ during photolysis. These results demonstrate selectivity of the photoprobe for the NAD ϩ binding site and suggest that the peptide identified using the photoprobe is located in the NAD ϩ binding domain of the brain GDH isoproteins. Both sequencing and compositional analysis identified Glu 275 as the site of photoinsertion.

DISCUSSION
In the present work, we identified an adenine binding domain peptide of the NAD ϩ binding site of two GDH isoproteins from bovine brain using photoaffinity probe [ 32 P]2N 3 NAD ϩ and peptide analysis. [ 32 P]2N 3 NAD ϩ is a probe that, on photolysis, generates a very reactive nitrene that has the capacity of photoinserting into any residue. The data showing decreased photoinsertion by addition of NAD ϩ demonstrates that photoinsertion occurs only by the bound form of [ 32 P]2N 3 NAD ϩ . This indicates that proximity controls photoinsertion and that the residues modified are within the adenine binding domain. In addition, pre-photolysis followed by immediate addition of GDH isoproteins did not lead to covalent labeling (data not shown), eliminating the existence of any long lived chemically reactive intermediate that could be involved in covalently modifying enzymes. Saturation of photoinsertion at concentrations corresponding to that expected from the reversible binding affinities also strongly supports that the site being labeled is within the binding domain. Their selectivity and specificity have been utilized successfully to locate the specific base binding domains of nucleotide binding site peptides of many proteins (11, 14 -18).
The specificity of [ 32 P]2N 3 NAD ϩ and the utility of this probe as a good candidate for determining the NAD ϩ binding site were further supported by the following. First, in the absence of activating light, 2N 3 NAD ϩ is a substrate for the GDH isoproteins (Table I). The ability to mimic a native compound before photolysis has an advantage over determination of the enzyme function after modification. Second, the photoinsertion into GDH I and GDH II was saturated with [ 32 P]2N 3 NAD ϩ . Saturation of photoinsertion with [ 32 P]2N 3 NAD ϩ occurred at around the 40 M photoprobe with apparent K d values near 10 M for both GDH isoproteins (Fig. 1). Third, active site involvement was also supported by the effects of GTP and ADP on photoinsertion (Table II). These results, together with the enhancement of photoinsertion by glutarate, present evidence of active site labeling of GDH isoproteins with 2N 3 NAD ϩ .
To identify the site of photoinsertion, the photolabeled GDH isoproteins were digested with trypsin to produce peptides. Taking advantage of the existence of the two cis-hydroxyl groups on the ribose groups of the photoincorporated probe, the photolabeled peptides were isolated by using immobilized boronate column chromatography. It has been shown that immobilized boronates can be successfully used to fractionate adenine and pyridine nucleotides (33) and nucleosidyl peptides (10,34). By using a pH gradient elution, a highly purified radiolabeled peptide was obtained from each isoprotein, and the peptides exhibited an unusual UV spectrum with a maximum absorption of 262 nm and a shoulder near 278 nm, which verifies that an adduct of the [ 32 P]2N 3 NAD ϩ photoprobe is still covalently attached to the peptides. The boronate chromatography greatly reduces the possibility of any non-photolabeled peptide co-eluting on HPLC with the photolabeled peptide, which could give misleading results. The sequences identified in the present study correspond to residues 270 -289 of the amino acid sequence of well known bovine liver GDH (Table  III). On the basis of information obtained on the amino acid sequence determination and composition analysis of the isolated peptide, we suggest that the attachment site of [ 32 P]2N 3 NAD ϩ is Glu 275 .
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. The NADH binding site was proposed to be modified by an ATP analogue at Cys 319 (8), by a GMP probe at Met 169 and Tyr 262 (9), and by the adenosine analogue at Lys 420 and Tyr 190 (10). Similar results with discrepancies using classical chemical probes were also reported by the same research group to identify other regulatory sites within bovine liver GDH. For instance, the ADP binding site was proposed to be modified by two different AMP analogues at His 82 (35) and Arg 459 (36). These two residues are outside the catalytic cleft. The GTP binding site was also proposed to be modified by a fluorescent FSBA adenosine analogue at Tyr 262 (37). As indicated elsewhere (17), it is not clear why a hydrophobic adenosine-containing probe 5ЈFSB⑀A preferentially FIG. 4. Reversed-phase HPLC purification of tryptic peptides eluting from a boronate affinity column. The radioactive eluates (fractions 25ϳ27) from a boronate affinity column were loaded onto a C 18 reversed-phase column, eluted with an acetonitrile gradient (dashed line) at a flow rate of 0.5 ml/min, and monitored at 220 nm (solid line). One-minute fractions were collected. The solid line with closed circles represents radioactivity. Levels of 32 P were determined by liquid scintillation counting in aqueous phase. binds and react at a hydrophilic GTP binding site and does not react at the other adenosine binding sites. It seems, therefore, that the base moiety has not been effective at directing the site of modification by classical chemical probes. Chemically reactive probes, because of 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 studied. 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 (8,9).
Very recently, Stanley et al. (38) have reported that the hyperinsulinism-hyperammonemia syndrome is caused by mutations in GDH gene that affect enzyme sensitivity to GTPinduced inhibition. The mutations identified in the patients with hyperinsulinism and hyperammonemia (38) lie exactly within a sequence of 15 amino acids that we previously suggested contains the GTP binding site of the brain GDH isoproteins (24). On the other hand, the location of the mutations on GDH in those mutations are quite distinct from the GTP binding site identified by using the classical chemical probe (37). These results prove selectivity and specificity of the photoaffinity probe as a valid active site probe.
The crystal structure of Clostridium symbiosum GDH has been aligned with the primary sequence of the bovine liver GDH (28,39). 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 (40,41). The primary sequence of bovine liver GDH also suggests the presence of a ␤-␣-␤ motif corresponding to the motif within the bacterial subunit. The photolabeled peptide of brain GDH isoproteins identified in this work is located within the proposed NAD ϩ binding domain of bovine liver GDH (42,43), and predicted to form a ␤-pleated sheet which constitutes one of the six strands of parallel sheet found in the NAD ϩ binding domain. This site is also near the active site portion of GDH as previously predicted (44). Therefore, this sequence is expected to interact with the adenine ring of the coenzyme.
The importance of Glu 275 in coenzyme binding can be further supported from some interesting information that the invariant, functional residue, Asp, is found in the ␤-pleated sheet region of four known dehydrogenases and is conservatively changed to Glu 275 in mammalian GDH (42). With these dehydrogenases, Asp is proposed to be involved in hydrogen bond formation with the O-2Ј atom of the ribose group which could not be occurring, due to charge repulsion, with NADP ϩ . This probably accounts for the inability of NADP ϩ to be a substrate for these dehydrogenases whereas it is a good substrate for mammalian GDH. This concept is supported by the observation that the equivalent position of Glu 275 in NADP ϩ -specific dihydrofolate reductase is replaced by a positively charged Arg residue. It is likely that the reason for the Glu replacement for Asp in GDH is to produce a different active site conformation that allows both NAD ϩ and NADP ϩ binding.
To our knowledge, comparison of the detailed structure of active sites and regulatory sites of any GDH isoproteins rarely has been reported. The work presented here clearly identifies the NAD ϩ binding site of the brain GDH isoproteins in the overall sequence.