scholarly journals The metabolism of S-nitrosothiols in the trypanosomatids: the role of ovothiol A and trypanothione

2003 ◽  
Vol 371 (1) ◽  
pp. 49-59 ◽  
Author(s):  
Ryan N. VOGT ◽  
Daniel J. STEENKAMP
Keyword(s):  
Reductase Activity ◽  
Order Rate Constant ◽  
Second Order ◽  
Formaldehyde Dehydrogenase ◽  
Thiyl Radicals ◽  
Order Rate ◽  
Rate Limiting Step ◽  
Low Levels ◽  
Rate Limiting

It has recently been established that nitrosoglutathione is the preferred substrate of the glutathione-dependent formaldehyde dehydrogenase from divergent organisms. Trypanosomatids produce not only glutathione, but also glutathionylspermidine, trypanothione and ovothiol A. The formaldehyde dehydrogenase activity of Crithidia fasciculata was independent of these thiols and extracts possessed very low levels of nitrosothiol reductase activity with glutathione or its spermidine conjugates as the thiol component. Although ovothiol A did not form a stable nitrosothiol, it decomposed the S-nitroso groups of nitrosoglutathione (GSNO) and dinitrotrypanothione [T(SNO)2] with second-order rate constants of 19.12M-1·s-1 and 8.67M-1·s-1 respectively. The reaction of T(SNO)2 with ovothiol A, however, accelerated to a rate similar to that seen with GSNO. Ovothiol A can act catalytically to decompose these nitrosothiols, although non-productive mechanisms exist. The catalytic phase of the reaction was dependent on the production of thiyl radicals, since it was abolished in the presence of 5,5-dimethyl-1-pyrroline-N-oxide and the formation of nitric oxide could be detected by means of the conversion of oxyhaemoglobin into methaemoglobin. The rate-limiting step in the catalytic process was the reduction of oxidized ovothiol species and, in this respect, T(SNO)2 is a more efficient substrate than GSNO. Trypanothione decomposed GSNO with a second-order rate constant of 0.786M-1·s-1 and the major nitrogenous end product changed from nitrite to ammonia as the ratio of thiol to nitrosothiol increased. The results indicate that ovothiol A acts in synergy with trypanothione in the decomposition of T(SNO)2.

scholarly journals Folding initiation sites and protein folding.

1999 ◽  
Vol 46 (3) ◽  
pp. 487-508 ◽  
Author(s):  
M Dadlez
Keyword(s):  
Protein Folding ◽  
Protein Fragments ◽  
Denatured State ◽  
Local Structures ◽  
Rate Limiting Step ◽  
Structural Preferences ◽  
Low Levels ◽  
Rate Limiting ◽  
Transition State Barrier

The paper discusses the role of local structural preferences of protein segments in the folding of proteins. First a short overview of the local, secondary structures detected in peptides, protein fragments, denatured proteins and early folding intermediates is given. Next the discussion of their role in protein folding is presented based on recent literature and data obtained in our laboratory. In conclusion it is pointed out that, during folding, local structures populated at low levels in denatured state may facilitate the crossing of the folding transition state barrier, and consequently accelerate the rate limiting step in folding. However, the data show that this effect does not follow simple rules.

scholarly journals NO Scavenging through Reductive Nitrosylation of Ferric Mycobacterium tuberculosis and Homo sapiens Nitrobindins

2020 ◽  
Vol 21 (24) ◽  
pp. 9395
Author(s):  
Giovanna De Simone ◽  
Alessandra di Masi ◽  
Chiara Ciaccio ◽  
Massimo Coletta ◽  
Paolo Ascenzi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rate Constant ◽  
Homo Sapiens ◽  
Mixing Time ◽  
Order Rate Constant ◽  
Order Rate ◽  
Rate Limiting Step ◽  
Reductive Nitrosylation ◽  
Ferrous Heme ◽  
Rate Limiting

Ferric nitrobindins (Nbs) selectively bind NO and catalyze the conversion of peroxynitrite to nitrate. In this study, we show that NO scavenging occurs through the reductive nitrosylation of ferric Mycobacterium tuberculosis and Homo sapiens nitrobindins (Mt-Nb(III) and Hs-Nb(III), respectively). The conversion of Mt-Nb(III) and Hs-Nb(III) to Mt-Nb(II)-NO and Hs-Nb(II)-NO, respectively, is a monophasic process, suggesting that over the explored NO concentration range (between 2.5 × 10−5 and 1.0 × 10−3 M), NO binding is lost in the mixing time (i.e., NOkon ≥ 1.0 × 106 M−1 s−1). The pseudo-first-order rate constant for the reductive nitrosylation of Mt-Nb(III) and Hs-Nb(III) (i.e., k) is not linearly dependent on the NO concentration but tends to level off, with a rate-limiting step (i.e., klim) whose values increase linearly with [OH−]. This indicates that the conversion of Mt-Nb(III) and Hs-Nb(III) to Mt-Nb(II)-NO and Hs-Nb(II)-NO, respectively, is limited by the OH−-based catalysis. From the dependence of klim on [OH−], the values of the second-order rate constant kOH− for the reductive nitrosylation of Mt-Nb(III)-NO and Hs-Nb(III)-NO were obtained (4.9 (±0.5) × 103 M−1 s−1 and 6.9 (±0.8) × 103 M−1 s−1, respectively). This process leads to the inactivation of two NO molecules: one being converted to HNO2 and another being tightly bound to the ferrous heme-Fe(II) atom.

Inhibition of the Thrombin-Thrombomodulin by a Covalent Antithrombin-Heparin.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2693-2693
Author(s):  
Maria C. Van Walderveen ◽  
Leslie R. Berry ◽  
Anthony K.C. Chan
Keyword(s):  
Rate Constants ◽  
Order Rate Constant ◽  
Second Order ◽  
The Novel ◽  
Constant Concentration ◽  
High Potency ◽  
Order Rate ◽  
Covalently Linked

Abstract Endothelial thrombin·thrombomodulin (IIa-TM) complex is capable of activating protein C anticoagulant. In vivo, TM can exist in two forms: one with a chondroitin sulfate (CS) (acidic) and one without (non-acidic). TM binding has been shown to reduce the rate of IIa inhibition by antithrombin (AT) in the presence of unfractionated heparin (UFH), especially when the TM possesses a glycosaminoglycan chain. We have recently developed a covalent antithrombin-heparin (ATH) with high potency for inhibition of IIa bound to surfaces. In the present study we examined the role of the CS chain in the inhibition of IIa-TM complex by the novel anticoagulant, ATH, in comparison to AT + UFH. For this study, discontinuous second order rate constant (k2) inhibition assays of IIa-TM were performed at 37°C. IIa was incubated with TM for 30 min and inhibited using either 20nM AT + varying concentrations (0–6000nM) of UFH or a constant concentration of 10nM ATH. Residual IIa activity was measured at 405nm after stopping the reaction using a solution of polybrene and chromogenic substrate (S-2238). Second order rate constants (mean +/− SEM; n≥5) were calculated from plots of IIa-TM activity vs. time and differences determined using two-sample t-tests. Comparison of the second order rate constants for inhibition of IIa, IIa-TM(−CS), or IIa-TM(+CS) by AT + UFH (2.015 × 108 +/− 7.079 × 106 M−1min−1, 1.633 × 108 +/− 8.922 × 106 M−1min−1, and 5.248 × 107 +/− 5.693 × 106 M−1min−1, respectively) showed that the presence of TM reduced the rate of inhibition (with respect to IIa alone; p=0.012, p<0.001, respectively). Moreover, the presence of a CS on the TM inhibited IIa neutralization to a greater degree than when the CS is absent (p<0.001). Furthermore, ATH was a much faster inhibitor of IIa, IIa-TM(−CS), and TM(+CS) (1.486 × 109 +/− 1.193 × 108 M−1min−1, 1.370 × 109 +/− 1.246 × 108 M−1min−1, and 8.869 × 108 +/− 1.246 × 108 M−1min−1, respectively) compared to AT + UFH (p<0.001). CS removal by chondroitin ABC lyase (ABCase) showed that the rate of IIa-TM(+CS) inhibition significantly increased for both AT + UFH and ATH, while no significant change due to ABCase treatment was found for the rate of inhibition of IIa-TM(−CS) by either AT + UFH or ATH. We speculate that during IIa-TM(+CS) inhibition by ATH, the covalently linked heparin moiety may be able to repel (rotate) the CS chain in order for ATH to interact with the IIa-TM complex. In the case of AT + UFH, lack of permanent UFH linkage to AT may allow the CS to displace the UFH chain from binding both AT and IIa. Steric hindrance by CS chains remains a significant factor since the k2 values were lower for the AT + UFH or ATH reactions with IIa-TM(+CS) compared to those with IIa-TM(−CS). Given the rapid reaction rate with free or bound IIa, ATH significantly limits availability of IIa for coagulant or anticoagulant functions.

scholarly journals The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase

1991 ◽  
Vol 275 (2) ◽  
pp. 335-339 ◽  
Author(s):  
H C Hawkins ◽  
R B Freedman
Keyword(s):  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Native Enzyme ◽  
Liver Protein ◽  
Thiol Groups ◽  
Protein Disulphide Isomerase ◽  
Order Rate ◽  
Reactive Groups

1. The number of reactive thiol groups in mammalian liver protein disulphide-isomerase (PDI) in various conditions was investigated by alkylation with iodo[14C]acetate. 2. Both the native enzyme, as isolated, and the urea-denatured enzyme contained negligible reactive thiol groups; the enzyme reduced with dithiothreitol contained two groups reactive towards iodoacetic acid at pH 7.5, and up to five reactive groups were detectable in the reduced denatured enzyme. 3. Modification of the two reactive groups in the reduced native enzyme led to complete inactivation, and the relationship between the loss of activity and the extent of modification was approximately linear. 4. Inactivation of PDI by alkylation of the reduced enzyme followed pseudo-first-order kinetics; a plot of the pH-dependence of the second-order rate constant for inactivation indicated that the essential reactive groups had a pK of 6.7 and a limiting second-order rate constant at high pH of 11 M-1.s-1. 5. Since sequence data on PDI show the presence within the polypeptide of two regions closely similar to thioredoxin, the data strongly indicate that these regions are chemically and functionally equivalent to thioredoxin. 6. The activity of PDI in thiol/disulphide interchange derives from the presence of vicinal dithiol groups in which one thiol group of each pair has an unusually low pK and high nucleophilic reactivity at physiological pH.

Adenoviral expression of 15-lipoxygenase-1 in rabbit aortic endothelium: role in arachidonic acid-induced relaxation

2007 ◽  
Vol 292 (2) ◽  
pp. H1033-H1041 ◽  
Author(s):  
Nitin T. Aggarwal ◽  
Blythe B. Holmes ◽  
Lijie Cui ◽  
Helena Viita ◽  
Seppo Yla-Herttuala ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Rabbit Aorta ◽  
Epoxyeicosatrienoic Acid ◽  
Aortic Endothelial Cells ◽  
Aortic Endothelium ◽  
Rate Limiting Step ◽  
Immunohistochemical Studies ◽  
Rate Limiting

Endothelium-dependent vasorelaxation of the rabbit aorta is mediated by either nitric oxide (NO) or arachidonic acid (AA) metabolites from cyclooxygenase (COX) and 15-lipoxygenase (15-LO) pathways. 15-LO-1 metabolites of AA, 11,12,15-trihydroxyeicosatrienoic acid (THETA), and 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) cause concentration-dependent relaxation. We tested the hypothesis that in the 15-LO pathway of AA metabolism, 15-LO-1 is sufficient and is the rate-limiting step in inducing relaxations in rabbit aorta. Aorta and rabbit aortic endothelial cells were treated with adenoviruses containing human 15-LO-1 cDNA (Ad-15-LO-1) or β-galactosidase (Ad-β-Gal). Ad-15-LO-1-transduction increased the expression of a 75-kDa protein corresponding to 15-LO-1, detected by immunoblotting with an anti-human15-LO-1 antibody, and increased the production of HEETA and THETA from [14C]AA. Immunohistochemical studies on Ad-15-LO-1-transduced rabbit aorta showed the presence of 15-LO-1 in endothelial cells. Ad-15-LO-1-treated aortic rings showed enhanced relaxation to AA (max 31.7 ± 3.2%) compared with Ad-β-Gal-treated (max 12.7 ± 3.2%) or control nontreated rings (max 13.1 ± 1.6%) ( P < 0.01). The relaxations in Ad-15-LO-1-treated aorta were blocked by the 15-LO inhibitor cinnamyl-3,4-dihydroxy-a-cyanocinnamate. Overexpression of 15-LO-1 in the rabbit aortic endothelium is sufficient to increase the production of the vasodilatory HEETA and THETA and enhance the relaxations to AA. This confirms the role of HEETA and THETA as endothelium-derived relaxing factors.

Kinetics of reconstitution of apotyrosinase by copper

1990 ◽  
Vol 68 (2) ◽  
pp. 476-479
Author(s):  
Donald C. Wigfield ◽  
Douglas M. Goltz
Keyword(s):  
Rate Constant ◽  
Copper Concentration ◽  
Copper Ions ◽  
Copper Ion ◽  
Order Rate Constant ◽  
First Order ◽  
Order Rate ◽  
Pseudo First Order ◽  
Rate Limiting ◽  
Kinetics Of

The kinetics of the reconstitution reaction of apotyrosinase with copper (II) ions are reported. The reaction is pseudo first order with respect to apoenzyme and the values of these pseudo first order rate constants are reported as a function of copper (II) concentration. Two copper ions bind to apoenzyme, and if the second one is rate limiting, the kinetically relevant copper concentration is the copper originally added minus the amount used in binding the first copper ion to enzyme. This modified copper concentration is linearly related to the magnitude of the pseudo first order rate constant, up to a copper concentration of 1.25 × 10−4 M (10-fold excess), giving a second order rate constant of 7.67 × 102 ± 0.93 × 102 M−1∙s−1.Key words: apotyrosinase, copper, tyrosinase.

Termolecular ion–molecule reactions involving C3H5+

1970 ◽  
Vol 48 (22) ◽  
pp. 3549-3553 ◽  
Author(s):  
A. G. Harrison ◽  
A. A. Herod
Keyword(s):  
Kinetic Energy ◽  
Molecular Size ◽  
Order Rate Constant ◽  
Second Order ◽  
Rate Coefficients ◽  
Third Order ◽  
Order Rate ◽  
Ion Molecule Reactions ◽  
Similar Dependence ◽  
Ion Kinetic Energy

The reaction of C3H5+ with C2D4 to produce C5H5D4+ is shown to be second order in C2D4. The rate coefficients are in the range 10−24 to 10−25 cm6 molecule−2 s−1 but decrease markedly with increasing ion kinetic energy. This decrease reflects the effect of the ion kinetic energy on the lifetime of the initial collision complex. Small differences in rate coefficients are observed depending on the source of the C3H5+ ion but these are insufficient to distinguish between possibly different ionic structures. The reaction of C3H5+ with C2H3F forms C5H7+ in a reaction second order in C2H3F. The rate coefficients are also in the range 10−24 to 10−25 cm6 molecule−1 s−1 and show a similar dependence on ion kinetic energy. These high third order rate constants are compared with data for other termolecular reactions and are shown to be consistent with the effect of molecular size on the third order rate constant.

Oxidation of bisphenol A by permanganate: reaction kinetics and removal of estrogenic activity

2013 ◽  
Vol 67 (8) ◽  
pp. 1867-1872 ◽  
Author(s):  
Jingjing Yang ◽  
Gang Wen ◽  
Ji Zhao ◽  
Xiaoling Shao ◽  
Jun Ma
Keyword(s):  
Bisphenol A ◽  
Reaction Kinetics ◽  
Estrogenic Activity ◽  
Complete Removal ◽  
Order Rate Constant ◽  
Second Order ◽  
Specific Rate ◽  
Order Rate ◽  
Ph Dependency ◽  
Ph Range

The kinetics for reaction between bisphenol A (BPA) and permanganate was examined over pH range of 5.0–9.9 and the estrogenic activity of aqueous BPA solution after oxidation was assessed by yeast two-hybrid assay. Reaction kinetics follows the second-order rate law, with the apparent second-order rate constant of 15.1 ± 1.1 M−1s−1 at pH 6.0 and 25°C and the activation energy of 48.7 kJ/mol. The kinetics exhibits pH dependency and the specific rate constants related to speciation of BPA are 50 ± 28 M−1s−1, 9.6 (±0.6) × 103 M−1s−1 and 1.4 (±0.1) × 104 M−1s−1 for BPA, BPA− and BPA2−, respectively. The results of the estrogenic/antiestrogenic activity test show that there is a hysteresis for the removal of estrogenic activity of aqueous BPA solution at pH 7.3. Removal of BPA is completed in 10 min, but complete removal of estrogenic activity requires a further 20 min.

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