Interaction of the photoaffinity label 8‐azido‐ADP with glutamate dehydrogenase

Koberstein, Rudolf; Cobianchi, L.; Sund, Horst

doi:10.1016/0014-5793(76)80277-3

Cited by 38 publications

(14 citation statements)

References 9 publications

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“…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 8N 3 ADP and ADP induced similar effects on the structure of GDH (31). This is consistent with the results in Fig.…”

Section: Discussionsupporting

confidence: 82%

“…1 shows, both ADP and 8N 3 ADP activated the activities of the GDH isoproteins, although maximal activity with 8N 3 ADP was about 75% of maximal ADPstimulated 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, 8N 3 ADP, is able to elicit the similar biological effects on GDH isoproteins as the natural nucleotide, ADP.…”

Section: Activation Of Gdh Isoproteins By Adp and 8n 3 Adp-thesupporting

confidence: 83%

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Photoaffinity Labeling of Brain Glutamate Dehydrogenase Isoproteins with an Azido-ADP

Cho

Yoon

1999

Journal of Biological Chemistry

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The ADP binding site within two types of bovine brain glutamate dehydrogenase isoproteins (GDH I and GDH II) was identified using photoaffinity labeling with [ Glutamate dehydrogenase (GDH 1 ; EC 1.4.1.3) is a family of enzymes that catalyze the reversible deamination of L-glutamate to 2-oxoglutarate using NAD ϩ , NADP ϩ , 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 Cys 319 (9), by a GMP probe at Met 169 and Tyr 262(10), and by the adenosine analogue at Lys 420 and Tyr 190 (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. Stanley et al. (23) have reported that the hyperinsulinism-hyperammonemia syndrome is caused by mutat...

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Section: Discussionsupporting

confidence: 82%

Section: Activation Of Gdh Isoproteins By Adp and 8n 3 Adp-thesupporting

confidence: 83%

Photoaffinity Labeling of Brain Glutamate Dehydrogenase Isoproteins with an Azido-ADP

Cho

Yoon

1999

Journal of Biological Chemistry

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“…The effect of various nucleotides and their photoaffinity analogs on the reductive amination of α‐ketoglutarate (reverse reaction) has been reported previously from this laboratory and elsewhere [11,12,20]. The presence of 100 µ m GTPγBP inhibited GDH activity by at least 80%.…”

Section: Resultsmentioning

confidence: 60%

Orientation of GTP and ADP within their respective binding sites in glutamate dehydrogenase

Rajagopalan

Watt

Haley

1999

European Journal of Biochemistry

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Previous studies have identified the guanine and adenine binding domains of the GTP and ADP binding sites of GDH. In this study the peptide sequences within or near to the terminal phosphate-binding domains of the GTP and ADP binding sites of bovine liver glutamate dehydrogenase (GDH) were identified using photoaffinity labeling with the benzophenone nucleotide derivatives, [g-32 P]GTPgBP and [g-32 P]ATPgBP. Without activating light, GTPgBP exhibited inhibiting effects on the GDH reaction similar to GTP; ATPgBP, as expected, produced activating effects similar to those of ADP. Photoinsertion into GDH by both probes exhibited saturation effects in agreement with the respective kinetic effects. Specificity of labeling was supported by specific and effective reduction of photoinsertion of [g-32 P]GTPgBP and [g-32 P]ATPgBP into GDH by GTP and ADP, respectively. Using a combination of immobilized Fe 3+ -chelate affinity chromatography and reversed-phase HPLC, photolabeled peptides located within or near the phosphate-binding domains of the GTP and ADP sites were isolated. Sequence analysis showed that GTPgBP primarily modified a peptide near the middle of the GDH sequence, Asn135±Lys143 and Glu290±Lys295. However, ATPgBP modified a single peptide corresponding to the sequence Met411±Arg419 near the C-terminal domain. Using these results and the data from the previously identified base-binding domain peptides the orientation of GTP and ADP within their respective binding sites in the catalytic cleft of GDH is proposed and explained on the basis of a proposed three-dimensional schematic model structure derived from the bacterial enzyme.Keywords: allosteric; enzyme regulation; nucleotide binding; photoaffinity; protein structure.Bovine liver glutamate dehydrogenase (GDH) plays a role in regulating the levels of ammonia and glutamate in the central nervous system and integrates carbon and nitrogen metabolism via the tricarboxylic acid cycle. GDH catalyzes the forward and reverse reaction of oxidatively deaminating l-glutamate into a-ketoglutarate and NH 4 + and utilizes the coenzymes NAD + and NADP + as electron acceptors [1].Bovine liver GDH is an NAD + /NADP + utilizing enzyme that consists of six identical subunits (56 kDa each) for which no crystal structure is available. However, the primary sequence has been published [2], and the crystal structure of the enzyme from Clostridium symbioses has been determined to 1.6 A Ê resolution [3]. The two enzymes are structurally similar as inferred from their sequences and electron micrographs [4±6]. GDH activity is regulated by numerous factors including pH, aggregation state, a variety of inhibitors and activators [7,8] but primarily by inhibition with GTP and activation by ADP. In addition, NADH, a substrate for the reverse reaction, has also been proposed to inhibit the forward reaction [9,10].Various nucleotide photoaffinity probes have been used to identify the base-binding domains of the NAD + and GTP sites [5,11±13] and to localize both domains within the proposed cat...

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“…Thefindings here reported are applicable to the photolysis of other azidopurine derivatives (10,16,17,24,25,43) and in fact, provide general product guidelines for those using azido-substituted purines as photoaffinity labels. NX = NH2 (Table I).…”

Section: Photolysismentioning

confidence: 94%

Active Cytokinins

et al. 1979

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Four series of azidopurines have been synthesized and tested for cytokinin activity in the tobacco callus bioassay: 2-and 8-azido-N6-benzyladenines, -N -(A2-isopentenyl)adenines, and -zeatins, and N -(2-and 4-azidobenzyl)adenines. The compounds having 2-azido substitution on the adenine ring are as active as the corresponding parent compounds, while those with 8-azido substitution are about 10 or more times as active. The 8-azidozeatin, which is the most active cytokinin observed, exhibited higher than minimal detectable activity at 1.2 x 10-5 micromolar, the lowest concentration tested. The shape of the growth curve indicates that even a concentration as low as 5 x 10-6 micromolar would probably be effective. By comparison, the lowest active concentration ever reported for zeatin has been 5 x 10-5 micromolar, representing a sensitivity rarely attained.All of the azido compbunds have been submitted to photolysis in aqueous ethanol, and the photoproducts have been detected and identified by low and high resolution mass spectrometry. They are rationalized as products of abstraction and insertion reactions of the intermediate nitrenes. The potential of the major released products as cytokinins was also assessed by bioassay. 2-Azido-N6-(A2-isopentenyl)adenine competed with 114CIki-netin for the cytokinin-binding protein isolated from wheat germ. When the azido compound was photolysed in the presence of this protein, its attachment effectively blocked the binding of 114Clkinetin.Extensive studies of structure-activity relationships for cytokinins have led to a well-defined concept of the chemical nature of '

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Interaction of the photoaffinity label 8‐azido‐ADP with glutamate dehydrogenase

Cited by 38 publications

References 9 publications

Photoaffinity Labeling of Brain Glutamate Dehydrogenase Isoproteins with an Azido-ADP

Photoaffinity Labeling of Brain Glutamate Dehydrogenase Isoproteins with an Azido-ADP

Orientation of GTP and ADP within their respective binding sites in glutamate dehydrogenase

Active Cytokinins

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