S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase induces formation of C150-C154 intrasubunit disulfide bond in the active site of the enzyme

The receptor for acetylcholine in the subsynaptic membrane of the electroplax of Electrophorus electricus is a protein with a disulfide bond in the vicinity of the active site. This disulfide can be reduced and reoxidized with concomitant inhibition and restoration of the response to acetylcholine and other monoquaternary ammonium-depolarizing agents. Conversely, the bisquaternary hexamethonium, normally a competitive inhibitor, causes depolarization, and the activity of decamethonium is increased following reduction of the disulfide. The reduced receptor can be alkylated by various maleimide derivatives and is then no longer reoxidizable. Some quaternary ammonium maleimide derivatives act as affinity labels of the reduced receptor, alkylating it at a rate three orders of magnitude faster then do uncharged maleimide derivatives. Other types of potential affinity labels also react only with the reduced receptor and the resulting covalently attached quaternary ammonium moieties interact with the active site, strongly activating the receptor. These results suggest a model for the active site and its transitions in which an activator such as acetylcholine bridges between a negative subsite and a hydrophobic subsite in the vicinity of the disulfide, causing an altered conformation around the negative subsite and a decrasee of a few angstroms in the distance between the two subsites.

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Disulfide bond formation between the active-site thiol and one of the several free thiol groups of chymopapain

Biochemistry ◽

10.1021/bi00370a024 ◽

1986 ◽

Vol 25 (22) ◽

pp. 6895-6900 ◽

Cited By ~ 7

Author(s):

Ibolya Korodi ◽

Bence Asboth ◽

Laszlo Polgar

Keyword(s):

Disulfide Bond ◽

Active Site ◽

Bond Formation ◽

Disulfide Bond Formation ◽

Thiol Groups ◽

Free Thiol

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Key Role for DsbA in Cell-to-Cell Spread ofShigella flexneri, Permitting Secretion of Ipa Proteins into Interepithelial Protrusions

Infection and Immunity ◽

10.1128/iai.68.11.6449-6456.2000 ◽

2000 ◽

Vol 68 (11) ◽

pp. 6449-6456 ◽

Cited By ~ 27

Author(s):

Jun Yu ◽

Bryn Edwards-Jones ◽

Olivier Neyrolles ◽

J. Simon Kroll

Keyword(s):

Confocal Microscopy ◽

Disulfide Bond ◽

Active Site ◽

Shigella Flexneri ◽

Wild Type ◽

Pathogenic Potential ◽

Previous Demonstration ◽

Intercellular Spread ◽

Cell Spread

ABSTRACT DsbA, a disulfide bond catalyst, is necessary for realization of the pathogenic potential of Shigella flexneri. Sh42, a mutant strain differing from wild-type M90TS solely because it expresses nonfunctional DsbA33G (substitution for 33C at the active site), secreted less IpaB and IpaC than M90TS in response to various stimuli in vitro. A kinetic study demonstrated that Sh42 responded more slowly to Congo red than M90TS. By modulating relative concentrations of functional and nonfunctional DsbA within bacteria, functional enzyme has been shown to be necessary for intercellular spread. By confocal microscopy, M90TS dividing in protrusions was shown to secrete Ipa proteins from the septation furrow, anticipating lysis of protrusions, while Sh42 showed minimal Ipa secretion in this location. In the light of a previous demonstration that DsbA is not necessary for entry of epithelial cells, we conclude that a role in virulence of this disulfide bond catalyst lies in facilitating secretion of Ipa proteins specifically within epithelial protrusions, in turn allowing cell-to-cell spread of S. flexneri.

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Formation of a covalent complex between methylguanine methyltransferase and DNA via disulfide bond formation between the active site cysteine and a thiol-containing analog of guanine

Nucleic Acids Research ◽

10.1093/nar/25.9.1795 ◽

1997 ◽

Vol 25 (9) ◽

pp. 1795-1801 ◽

Cited By ~ 14

Author(s):

S. R. Paalman ◽

N. D. Clarke ◽

D. M. Noll

Keyword(s):

Disulfide Bond ◽

Active Site ◽

Bond Formation ◽

Disulfide Bond Formation ◽

Active Site Cysteine ◽

Methylguanine Methyltransferase ◽

Covalent Complex

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Replacement of the Active-Site Cysteine Residues of DsbA, a Protein Required for Disulfide Bond Formation in vivo

Biochemistry ◽

10.1021/bi00173a038 ◽

1994 ◽

Vol 33 (7) ◽

pp. 1907-1914 ◽

Cited By ~ 39

Author(s):

Andre Zapun ◽

Leanne Cooper ◽

Thomas E. Creighton

Keyword(s):

Disulfide Bond ◽

Active Site ◽

Bond Formation ◽

Cysteine Residues ◽

Disulfide Bond Formation ◽

Active Site Cysteine

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Structural studies on the catalytic mechanism of Diaminopropionate ammonia lyase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314081789 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1822-C1822

Author(s):

Geeta Deka ◽

Shveta Bisht ◽

H.S. Savithri ◽

M.R.N Murthy

Keyword(s):

Reaction Mechanism ◽

Disulfide Bond ◽

Active Site ◽

Catalytic Mechanism ◽

Biochemical Characterization ◽

Catalytic Efficiency ◽

Peptide Antibiotics ◽

Type Ii ◽

Structural Studies ◽

Fold Type

Diaminopropionate ammonia lyase (DAPAL) is a non-stereo specific fold-type II pyridoxal 5' phosphate (PLP) dependent enzyme that catalyzes the conversion of both D/L isoforms of the nonstandard amino acid Diaminopropionate (DAP) to pyruvate and ammonia. DAP is important for the synthesis of nonribosomal peptide antibiotics such as viomycin and capreomycin. Earlier structural studies on EcDAPAL bound to a reaction intermediate (aminoacrylate) suggested that the enzyme follows a two base mechanism, where Asp120 and Lys77 function as general bases to abstract proton from D-DAP and L-DAP respectively. A novel disulfide was observed near the active site, although its functional significance was not clear. In the present study, structural and biochemical characterization of active site mutants Asp120 (Asp120Asn/Ser/Thr/Cys) and Lys77 (Lys77His/ Thr/Ala/Val) of EcDAPAL has been carried out. Reduction of catalytic efficiency (Kcat/Km) of D120N EcDAPAL for D-DAP by 140 fold and presence of the uncatalyzed ligand at the active site in the crystal structure suggested that Asp120 indeed abstracts proton from D-DAP. Lys77, which was speculated to be important for proton abstraction from L DAP, however seemed to be crucial for PLP binding only. Presence of non-covalently bound PLP in the L77W mutant and occurence of both the ketoenamine, enolimine forms of internal aldimine in L77R mutant provided an in depth insight into the unique chemistry of internal aldimine formation in PLP dependent enzymes. To investigate the role of the novel disulfide bond near the active site, C265 and C291 were mutated to Serine. Studies on these mutants show that this disulfide bond gives additional stability to the protein and might regulate the entry of substrates to the active site. Thus, these studies provide deeper insights into the reaction mechanism of EcDAPAL, representing the overall reaction mechanism followed by several other fold-type II PLP pendent enzymes.

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