scholarly journals Mammalian Spermidine Synthase—Identification of Cysteine Residues and Investigation of the Putrescine Binding Site—

2004 ◽  
Vol 27 (9) ◽  
pp. 1327-1332 ◽  
Author(s):  
Hitomi Goda ◽  
Toshiko Watanabe ◽  
Noboru Takeda ◽  
Masaki Kobayashi ◽  
Makiko Wada ◽  
...  
Keyword(s):  
Binding Site ◽  
Cysteine Residues ◽  
Spermidine Synthase

scholarly journals The human SNARE protein Ykt6 mediates its own palmitoylation at C-terminal cysteine residues

2004 ◽  
Vol 384 (2) ◽  
pp. 233-237 ◽  
Author(s):  
Michael VEIT
Keyword(s):  
Fusion Protein ◽  
Binding Site ◽  
Covalent Attachment ◽  
Receptor Protein ◽  
Cysteine Residues ◽  
Snare Protein ◽  
Attachment Protein ◽  
Present Evidence ◽  
Protein Receptor ◽  
Protein Attachment

The yeast SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) protein Ykt6 was shown to mediate palmitoylation of the fusion factor Vac8 in a reaction essential for the fusion of vacuoles. Here I present evidence that hYkt6 (human Ykt6) has self-palmitoylating activity. Incubation of recombinant hYkt6 with [3H]Pal-CoA ([3H]palmitoyl-CoA) leads to covalent attachment of palmitate to C-terminal cysteine residues. The N-terminal domain of human Ykt6 contains a Pal-CoA binding site and is required for the reaction.

scholarly journals Two conformationally vicinal thiols at the active site of Leishmania donovani adenosine kinase

1996 ◽  
Vol 316 (2) ◽  
pp. 439-445 ◽  
Author(s):  
Tapan K. BAGUI ◽  
Mallika GHOSH ◽  
Alok K. DATTA
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Leishmania Donovani ◽  
Marked Change ◽  
Adenosine Kinase ◽  
Inactivation Kinetics ◽  
Cysteine Residues ◽  
Schematic Model ◽  
Hydrophobic Domain ◽  
Modified Enzyme

Inactivation of adenosine kinase (Adk) from Leishmania donovani correlates with the modification of two conformationally vicinal cysteine residues. In contrast, Adk from hamster liver, despite being sensitive to monothiol-blocking reagents, was insensitive to dithiol modifiers. Inactivation kinetics and substrate-protection studies along with double-modification experiments successively with N-ethylmaleimide in the presence of Ado and sodium m-arsenite–2,3-dimercaptopropanol or diazenedicarboxylic acid bis-N,N´-dimethylamide supported assignment of the two thiols at the Ado-binding site. Cystine bridge formation impaired the ability of the modified enzyme to bind to the substrate. Tryptophan fluorescence of the enzyme was quenched after modification by dithiol-blocking reagents with concomitant loss of activity. However, treatment of the enzyme with methylmethanethiosulphonate (MMTS) led to complete inactivation without a marked change in protein fluorescence. Ado protected both fluorescence and catalytic activity against inactivation by both MMTS and dithiol-blocking reagents. Stern–Volmer quenching analysis of the native and Ado-complexed enzyme suggested that, of the four tryptophan residues, at least one is located at or near the active site. Furthermore quenching constants of native, Ado-complexed and dithiol-modified enzyme in the presence of either acrylamide or KI indicated spatial proximity of tryptophan and two cysteine residues within the hydrophobic domain of the Ado-binding site. Taken together the results suggest important function(s) for the cysteine residue(s). A schematic model is proposed to explain the inactivation of the enzyme by both monothiol- and dithiol-blocking reagents.

Arginine and cysteine residues in the ligand binding site of alpha 2- adrenergic receptors

Life Sciences ◽  
1985 ◽  
Vol 36 (4) ◽  
pp. 355-362 ◽  
Author(s):  
E. Mattens ◽  
S. Bottari ◽  
A. Vokaer ◽  
G. Vauguelin
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Adrenergic Receptors ◽  
Cysteine Residues ◽  
Ligand Binding Site

Highly reactive cysteine residues are part of the substrate binding site of mammalian dipeptidyl peptidases III

2004 ◽  
Vol 36 (3) ◽  
pp. 434-446 ◽  
Author(s):  
Marija Abramić ◽  
Šumski Šimaga ◽  
Maja Osmak ◽  
Lipa Čičin-Šain ◽  
Bojana Vukelić ◽  
...  
Keyword(s):  
Binding Site ◽  
Substrate Binding ◽  
Cysteine Residues ◽  
Substrate Binding Site ◽  
Dipeptidyl Peptidases

RirA of Dinoroseobacter shibae senses iron via a [3Fe–4S]1+ cluster co-ordinated by three cysteine residues

2020 ◽  
Vol 477 (1) ◽  
pp. 191-212
Author(s):  
Maren Behringer ◽  
Lisa Plötzky ◽  
Dirk Baabe ◽  
Marc-Kevin Zaretzke ◽  
Peter Schweyen ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Growth Conditions ◽  
Cysteine Residues ◽  
Mobility Shift ◽  
Paramagnetic Resonance ◽  
Dinoroseobacter Shibae ◽  
Electro Mobility

In the marine bacterium, Dinoroseobacter shibae the transcription factor rhizobial iron regulator A (RirA) is involved in the adaptation to iron-limited growth conditions. In vitro iron and sulfide content determinations in combination with UV/Vis and electron paramagnetic resonance (EPR) spectroscopic analyses using anaerobically purified, recombinant RirA protein suggested a [3Fe–4S]1+ cluster as a cofactor. In vivo Mössbauer spectroscopy also corroborated the presence of a [3Fe–4S]1+ cluster in RirA. Moreover, the cluster was found to be redox stable. Three out of four highly conserved cysteine residues of RirA (Cys 91, Cys 99, Cys 105) were found essential for the [3Fe–4S]1+ cluster coordination. The dimeric structure of the RirA protein was independent of the presence of the [3Fe–4S]1+ cluster. Electro mobility shift assays demonstrated the essential role of an intact [3Fe–4S]1+ cluster for promoter binding by RirA. The DNA binding site was identified by DNase I footprinting. Mutagenesis studies in combination with DNA binding assays confirmed the promoter binding site as 3′-TTAAN10AATT-5′. This work describes a novel mechanism for the direct sensing of cellular iron levels in bacteria by an iron-responsive transcriptional regulator using the integrity of a redox-inactive [3Fe–4S]1+ cluster, and further contributes to the general understanding of iron regulation in marine bacteria.

Antithrombin-mediated Inhibition of Factor Vlla-Tissue Factor Complex by the Synthetic Pentasaccharide Representing the Heparin Binding Site to Antithrombin

1996 ◽  
Vol 76 (01) ◽  
pp. 005-008 ◽  
Author(s):  
Jean Claude Lormeau ◽  
Jean Pascal Herault ◽  
Jean Marc Herbert
Keyword(s):  
Binding Site ◽  
Tissue Factor ◽  
Residual Activity ◽  
Coagulation Factors ◽  
Heparin Binding ◽  
Experimental Conditions ◽  
First Order ◽  
Heparin Binding Site ◽  
Coagulant Activity

SummaryWe examined the effect of the synthetic pentasaccharide representing the minimal binding site of heparin to antithrombin on the antithrombin-mediated inactivation of factor Vila bound to tissue factor. This effect was compared to the effect of unfractionated heparin. Using purified recombinant human coagulation factors and either a clotting or an amidolytic assay for the determination of the residual activity of factor Vila, we showed that the pentasaccharide was an efficient antithrombin-dependent inhibitor of the coagulant activity of tissue factor-factor Vila complex. In our experimental conditions, assuming a mean MW of 14,000 for heparin, the molar pseudo-first order rate constants for ATIII-mediated FVIIa inhibition by ATIII-binding heparin and by the synthetic pentasaccharide were found to be similar with respective values of 104,000 ± 10,500 min-1 and 112,000 ± 12,000 min-1 (mean ± s.e.m., n = 3)

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