Dynamical structure of glutamate dehydrogenase as monitored by tryptophan phosphorescence

Cioni, Patrizia; Strambini, Giovanni B.

doi:10.1016/0022-2836(89)90453-1

Cited by 43 publications

(30 citation statements)

References 40 publications

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“…ATP decreases the average phosphorescence lifetime from 2.8 to 1.9 ms. This lifetime decrease indicates an enhanced flexibility of the polypeptide chain surrounding the chromophore (35,49,50). Thus, the addition of ATP to Nd-F 1 induces conformational changes of the protein, resulting in a more flexible environment for the Nterminal segment of the ⑀-subunit, where Trp is located at position 4 (6).…”

Section: Methodsmentioning

confidence: 99%

“…1 The abbreviations used are: F 1 (x,y), F 1 containing x mol of ANP at noncatalytic sites and y mol of ANP at catalytic sites per mol of enzyme; F 1 , soluble part of the F-type H ϩ -ATPases; Nd-F 1 , nucleotide-depleted F 1 ; AMP-PNP, 5Ј-adenylyl-␤,␥-imidodiphosphate; HPLC, high pressure liquid chromatography. the indole nucleus to the flexibility of its surrounding matrix (34) has been extremely useful with respect to revealing subtle conformational changes induced in proteins by binding of substrates, coenzymes, inhibitors, or interacting macromolecules (35)(36)(37). We have recently used the phosphorescence of the sole tryptophan residue of the mitochondrial F 1 complex as an internal probe of the ⑀-subunit (38,39), and the lifetime () measurements have revealed conformational changes of the nucleotide-depleted enzyme as a consequence of Mg-ATP binding at low temperature.…”

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confidence: 99%

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Conformational Changes of the Mitochondrial F1-ATPase ∊-Subunit Induced by Nucleotide Binding as Observed by Phosphorescence Spectroscopy

Baracca

Gabellieri

Barogi

et al. 1995

Journal of Biological Chemistry

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Changes in conformation of the ⑀-subunit of the bovine heart mitochondrial F 1 -ATPase complex as a result of nucleotide binding have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas nucleoside triphosphate binding to the enzyme in the presence of Mg 2؉ increases the flexibility of the protein structure surrounding the chromophore, nucleoside diphosphate acts in an opposite manner, enhancing the rigidity of this region of the macromolecule. Such changes in dynamic structure of the ⑀-subunit are evident at high ligand concentration added to both the nucleotide-depleted F 1 (Nd-F 1 ) and the F 1 preparation containing the three tightly bound nucleotides (F 1 (2,1)). Since the effects observed are similar in both the F 1 forms, the binding to the low affinity sites must be responsible for the conformational changes induced in the ⑀-subunit. This is partially supported by the observation that the Trp lifetime is not significantly affected by adding an equimolar concentration of adenine nucleotide to Nd-F 1 . The effects on protein structure of nucleotide binding to either catalytic or noncatalytic sites have been distinguished by studying the phosphorescence emission of the F 1 complex prepared with the three noncatalytic sites filled and the three catalytic sites vacant (F 1 (3,0)). Phosphorescence lifetime measurements on this F 1 form demonstrate that the binding of Mg-NTP to catalytic sites induces a slight enhancement of the rigidity of the ⑀-subunit. This implies that the binding to the vacant noncatalytic site of F 1 (2,1) must exert the opposite and larger effect of enhancing the flexibility of the protein structure observed in both Nd-F 1 and F 1 (2,1). The observation that enhanced flexibility of the protein occurs upon addition of adenine nucleotides to F 1 (2,1) in the absence of Mg 2؉ provides direct support for this suggestion. The connection between changes in structure and the possible functional role of the ⑀-subunit is discussed.The ATPase (ATP synthase) is the enzyme responsible for ATP synthesis during oxidative phosphorylation in all energytransducing membranes. It is composed of two main parts: F 0

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Conformational Changes of the Mitochondrial F1-ATPase ∊-Subunit Induced by Nucleotide Binding as Observed by Phosphorescence Spectroscopy

Baracca

Gabellieri

Barogi

et al. 1995

Journal of Biological Chemistry

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“…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).…”

Section: Discussionmentioning

confidence: 99%

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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“…11 Phosphorescence lifetime represents a sensitive probe of local protein rigidity 12,13 and could be correlated to changes in rigidity triggered by binding of substrates or allosteric effectors. [14][15][16] Tryptophan phosphorescence at room temperature could provide information regarding the dynamic changes in protein structure occurring on a millisecond to second time scale. 12,[17][18][19][20][21] Besides an intrinsic lifetime of the triplet probe, the rate of migration of a phosphorescence quenching solute, such as acrylamide, into the proximity of the phosphorescent probe provides information on the local segmental flexibility and structural fluctuations.…”

Section: Introductionmentioning

confidence: 99%

“…The exceptionally rigid and solvent-inaccessible environment in the inner core of the macromolecule provides for a very long phosphorescence lifetime, up to two seconds. [11][12][13][14][15][16][17][18][19][20][21][22][23] Trp109 is connected to the active site via a rigid α-helical rod and to the subunit interface via a β-sheet that envelopes Trp109. 8 Therefore, the triplet lifetime of this residue could be used as a sensitive monitor of the conformational state in the polypeptide region that connects the active sites of neighboring subunits.…”

Section: Introductionmentioning

confidence: 99%

Influence of Subunit Interface Mutations on Kinetic and Dynamic Properties of Alkaline Phosphatase from E. coli

Šprung¹,

Bučević-Popović²,

Soldo³

et al. 2013

Croat. Chem. Acta

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Abstract. Mutations, replacing amino acids involved in the formation of hydrogen bonds between subunits of dimeric alkaline phosphatase, have been introduced. Influence of mutations on kinetic properties and structural stability of mutant enzymes was established. In addition, alterations in protein dynamic properties have been studied using room temperature phosphorescence. Kinetic properties of both mutant enzymes were virtually the same, differing from the wild type enzyme in the k cat value that was almost twice lower. Changes in protein dynamic properties of mutant proteins, compared to the wild type enzyme, did not parallel changes in kinetic properties suggesting that an alteration in the rigidity of the Trp109 environment is not responsible for the reduction of kinetic properties. Instead, combined kinetic and dynamic consequences of introduced mutations suggest that breaking of specific links, involved in transmission of conformational change, could be responsible for altered kinetic properties. (doi: 10.5562/cca2168)

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Dynamical structure of glutamate dehydrogenase as monitored by tryptophan phosphorescence

Cited by 43 publications

References 40 publications

Conformational Changes of the Mitochondrial F1-ATPase ∊-Subunit Induced by Nucleotide Binding as Observed by Phosphorescence Spectroscopy

Conformational Changes of the Mitochondrial F1-ATPase ∊-Subunit Induced by Nucleotide Binding as Observed by Phosphorescence Spectroscopy

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

Influence of Subunit Interface Mutations on Kinetic and Dynamic Properties of Alkaline Phosphatase from E. coli

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