The effect of N-bromosuccinimide on ferredoxin: NADP+ oxidoreductase

Hirasawa, Masakazu; Kleis-SanFrancisco, Susan; Proske, Paul A.; Knaff, David B.

doi:10.1016/0003-9861(95)90011-x

Cited by 3 publications

(3 citation statements)

References 38 publications

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“…The labeling with NBS induces tryptophan oxidation (19) which is also considered to be a common artifact of sample handling (28). Since we observed tryptophan oxidation even in the unmodified controls, the residues labeled with NBS were determined by comparing peak intensities of the modified and the unmodified peptides (29).…”

Section: Methodsmentioning

confidence: 99%

“…CBS proteins (1 mg/mL) were diluted in modification buffer and covalently labeled. Each labeling procedure − (Table S1 in the Supporting Information) was repeated three times. Reaction was quenched by buffer exchange using Zeba Desalt spin columns (ThermoFischer Scientific) with elution by 50 mM NH 4 HCO 3 .…”

Section: Methodsmentioning

confidence: 99%

“…With the exception of labeling with NBS, all other modification sites were identified by detection of labeled peptides that were not detected in unmodified controls (27); expected mass shifts for each reaction are shown in Supporting Information Table S1. The labeling with NBS induces tryptophan oxidation (19) which is also considered to be a common artifact of sample handling (28). Since we observed tryptophan oxidation even in the unmodified controls, the residues labeled with NBS were determined by comparing peak intensities of the modified and the unmodified peptides (29).…”

Section: Methodsmentioning

confidence: 99%

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Cross-Talk between the Catalytic Core and the Regulatory Domain in Cystathionine β-Synthase: Study by Differential Covalent Labeling and Computational Modeling

et al. 2010

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Cystathionine β-synthase (CBS) is a modular enzyme which catalyzes condensation of serine with homocysteine. Cross-talk between the catalytic core and the C-terminal regulatory domain modulates the enzyme activity. The regulatory domain imposes an autoinhibition action that is alleviated by S-adenosyl-l-methionine (AdoMet) binding, by deletion of the C-terminal regulatory module, or by thermal activation. The atomic mechanisms of the CBS allostery have not yet been sufficiently explained. Using pulse proteolysis in urea gradient and proteolytic kinetics with thermolysin under native conditions, we demonstrated that autoinhibition is associated with changes in conformational stability and with sterical hindrance of the catalytic core. To determine the contact area between the catalytic core and the autoinhibitory module of the CBS protein, we compared side-chain reactivity of the truncated CBS lacking the regulatory domain (45CBS) and of the full-length enzyme (wtCBS) using covalent labeling by six different modification agents and subsequent mass spectrometry. Fifty modification sites were identified in 45CBS, and four of them were not labeled in wtCBS. One differentially reactive site (cluster W408/W409/W410) is a part of the linker between the domains. The other three residues (K172 and/or K177, R336, and K384) are located in the same region of the 45CBS crystal structure; computational modeling showed that these amino acid side chains potentially form a regulatory interface in CBS protein. Subtle differences at CBS surface indicate that enzyme activity is not regulated by conformational conversions but more likely by different allosteric mechanisms.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cross-Talk between the Catalytic Core and the Regulatory Domain in Cystathionine β-Synthase: Study by Differential Covalent Labeling and Computational Modeling

et al. 2010

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Ferredoxin and Ferredoxin-Dependent Enzymes

Knaff

1996

Oxygenic Photosynthesis: The Light Reactions

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The role of aromatic and acidic amino acids in the electron transfer reaction catalyzed by spinach ferredoxin-dependent glutamate synthase

Hirasawa

Hurley

Salamon

et al. 1998

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Treatment of the ferredoxin-dependent, spinach glutamate synthase with N-bromosuccinimide (NBS) modifies 2 mol of tryptophan residues per mol of enzyme, without detectable modification of other amino acids, and inhibits enzyme activity by 85% with either reduced ferredoxin or reduced methyl viologen serving as the source of electrons. The inhibition of ferredoxin-dependent activity resulting from NBS treatment arises entirely from a decrease in the turnover number. Complex formation of glutamate synthase with ferredoxin prevented both the modification of tryptophan residues by NBS and inhibition of the enzyme. NBS treatment had no effect on the secondary structure of the enzyme, did not affect the Kms for 2-oxoglutarate and glutamine, did not affect the midpoint potentials of the enzyme's prosthetic groups and did not decrease the ability of the enzyme to bind ferredoxin. It thus appears that the ferredoxin-binding site(s) of glutamate synthase contains at least one, and possibly two, tryptophans. Replacement of either phenylalanine at position 65, in the ferredoxin from the cyanobacterium Anabaena PCC 7120, with a non-aromatic amino acid, or replacement of the glutamate at ferredoxin position 94, decreased the turnover number compared to that observed with wild-type Anabaena ferredoxin. The effect of the change at position 65 was quite modest compared to that at position 94, suggesting that an aromatic amino acid is not absolutely essential at position 65, but that glutamate 94 is essential for optimal electron transfer.

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The effect of N-bromosuccinimide on ferredoxin: NADP+ oxidoreductase

Cited by 3 publications

References 38 publications

Cross-Talk between the Catalytic Core and the Regulatory Domain in Cystathionine β-Synthase: Study by Differential Covalent Labeling and Computational Modeling

Cross-Talk between the Catalytic Core and the Regulatory Domain in Cystathionine β-Synthase: Study by Differential Covalent Labeling and Computational Modeling

Ferredoxin and Ferredoxin-Dependent Enzymes

The role of aromatic and acidic amino acids in the electron transfer reaction catalyzed by spinach ferredoxin-dependent glutamate synthase

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