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.

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.

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 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.

The Dephosphorylation of Adenosine 5′ -Triphosphate in a Binary and Ternary Zn2+ Complex

1974 ◽  
Vol 29 (11-12) ◽  
pp. 680-682 ◽  
Author(s):  
Peter Amsler ◽  
David Buisson ◽  
Helmut Sigel
Keyword(s):  
Rate Constant ◽  
Ternary Complex ◽  
Order Rate Constant ◽  
Reactive Species ◽  
Ph Optimum ◽  
First Order ◽  
Order Rate

The dephosphorylation of ATP was characterized by determining the dependence of the first-order rate constant on pH in the absence and presence of Zn2+ and together with Zn2+ and 2,2′-bipyridyl. The Zn2+-accelerated reaction passes through a pH optimum at about 8. The decrease in the rate at higher pH is due to the formation of Zn(ATP) (OH)3-; this species is relatively insensitive towards dephosphorylation. It is concluded that Zn(ATP)2- is the reactive species and that the interaction between N (7) and Zn2+ in this complex is crucial for its reactivity. In the presence of 2,2′-bipyridyl (Bipy) the ternary complex, Zn (Bipy) (ATP)2-, is formed which is rather stable towards dephosphorylation. It is suggested that the described effects of acceleration and inhibition are helpful for understanding the recycled processes in nature.

Effect of Medium on Acid-Catalyzed Decomposition of N-(Phenylazo)-Substituted Nitrogen Heterocycles

1999 ◽  
Vol 64 (10) ◽  
pp. 1654-1672 ◽  
Author(s):  
Miroslav Ludwig ◽  
Iva Bednářová ◽  
Patrik Pařík
Keyword(s):  
Rate Constant ◽  
Principal Component ◽  
Catalyst Particle ◽  
Pivalic Acid ◽  
Linear Regression Method ◽  
Catalytic Rate Constant ◽  
Rate Limiting Step ◽  
Acid Catalyzed ◽  
Rate Limiting ◽  
Kinetics Of

Four N-(phenylazo)-substituted saturated nitrogen heterocyclics were synthesized and their structure was confirmed by 1H and 13C NMR spectroscopy. The kinetics of their acid-catalyzed decomposition were studied at various concentrations of the catalyst (pivalic acid) in 40, 30, and 20% (v/v) aqueous ethanol at 25 °C. The values obtained for the observed rate constants were processed by the non-linear regression method according to the suggested kinetic models and by the method of principal component analysis (PCA). The interpretation of the results has shown that the acid-catalyzed decomposition of the heterocyclics under the conditions used proceeds by the mechanism of general acid catalysis, the proton being the dominant catalyst particle of the rate-limiting step. The decrease in the observed rate constant at higher concentrations of the catalyst was explained by the formation of a non-reactive complex composed of the undissociated acid and the respective N-(phenylazo)heterocycle. The effect of medium and steric effect of the heterocyclic moiety on the values of catalytic rate constant are discussed.

scholarly journals Coassembly of SecYEG and SecA Fully Restores the Properties of the Native Translocon

2018 ◽  
Vol 201 (1) ◽  
Author(s):  
Priya Bariya ◽  
Linda L. Randall
Keyword(s):  
Lipid Bilayers ◽  
Rate Constant ◽  
Apparent Rate Constant ◽  
Membrane Vesicles ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Secretory System ◽  
Rate Limiting

ABSTRACTIn all cells, a highly conserved channel transports proteins across membranes. InEscherichia coli, that channel is SecYEG. Many investigations of this protein complex have used purified SecYEG reconstituted into proteoliposomes. How faithfully do activities of reconstituted systems reflect the properties of SecYEG in the native membrane environment? We investigated by comparing threein vitrosystems: the native membrane environment of inner membrane vesicles and two methods of reconstitution. One method was the widely used reconstitution of SecYEG alone into lipid bilayers. The other was our method of coassembly of SecYEG with SecA, the ATPase of the translocase. For nine different precursor species we assessed parameters that characterize translocation: maximal amplitude of competent precursor translocated, coupling of energy to transfer, and apparent rate constant. In addition, we investigated translocation in the presence and absence of chaperone SecB. For all nine precursors, SecYEG coassembled with SecA was as active as SecYEG in native membrane for each of the parameters studied. Effects of SecB on transport of precursors faithfully mimicked observations madein vivo. From investigation of the nine different precursors, we conclude that the apparent rate constant, which reflects the step that limits the rate of translocation, is dependent on interactions with the translocon of portions of the precursors other than the leader. In addition, in some cases the rate-limiting step is altered by the presence of SecB. Candidates for the rate-limiting step that are consistent with our data are discussed.IMPORTANCEThis work presents a comprehensive quantification of the parameters of transport by the Sec general secretory system in the threein vitrosystems. The standard reconstitution used by most investigators can be enhanced to yield six times as many active translocons simply by adding SecA to SecYEG during reconstitution. This robust system faithfully reflects the properties of translocation in native membrane vesicles. We have expanded the number of precursors studied to nine. This has allowed us to conclude that the rate constant for translocation varies with precursor species.

DFP (Dilsopropylfluorophosphate) Studies on Molecular Mechanisms of Surface-Dependent Activation of Prekallikrein(PK) and Factor XII(Hageman Factor, HF)

1979 ◽  
Author(s):  
J.H. Griffin ◽  
G. Beretta
Keyword(s):  
Enzymatic Activity ◽  
Rate Constant ◽  
Active Sites ◽  
Molecular Mechanisms ◽  
Order Rate Constant ◽  
Factor Xii ◽  
Single Chain ◽  
Hageman Factor ◽  
Order Rate ◽  
Coagulant Activity

Previous 3H-DFP studies showed that HF binding to kaolin does not result in formation of new active sites. New studies show that single chain zymogen HF, in solution or surface bound, reacts slowly with DFP causing loss of coagulant activity and uptake of 1 mol DIP/mol HF with a 2nd order rate constant, k2=0.4 M-1 min-1. High MW kininogen (HMWK) did not affect the rate constant for this reaction. PK reacts with DFP causing loss of coagulant activity with k2=0.6 M-1min-1. These values are ~103lower than for the activated enzymes (160 and 500 M-1 min-1 for HFa and kallikrein respectively) and are similar to values for trypsinogen that exhibits weak enzymatic activity. HF, PK, HMWK, and kaolin were separately preincubated with 40mM DFP for 5 min to inhibit traces of active enzymes. Then, mixing of these reagents in the presence of 40mM DFP caused a burst of cleavage of 131I-HF and 125I-PK, the extent of which depended on the amount of HMWK. To avoid reciprocal proteolysis of HF and PK, “killed” zymogens (DIP-HF or DIP-PK formed by 48 hr incubation with 40mM DFP) were used in place of HF or PK. Controls showed that PK and DIP-PK were similarly cleaved by purified HFa. If DIP-131I-HF or DIP-125I-PK was substituted for HF or PK in mixtures of HF, PK, HMWK, and kaolin, no detectable cleavage occurred. These data allow that inherent activity of single chain forms of HF or PK may trigger-surface dependent reactions, but the vast majority of molecules are activated by reciprocal proteolysis between HF and PK with HMWK as a cofactor.

The mechanisms of hydrolysis of alkyl N-alkylthioncarbamate esters at 100 °C

2005 ◽  
Vol 83 (9) ◽  
pp. 1483-1491 ◽  
Author(s):  
Eduardo Humeres ◽  
Maria de Nazaré M. Sanchez ◽  
Conceição ML Lobato ◽  
Nito A Debacher ◽  
Eduardo P. de Souza
Keyword(s):  
Rate Constant ◽  
Order Rate Constant ◽  
Base Catalysis ◽  
Hydrolysis Rate ◽  
Negative Slope ◽  
General Base ◽  
Order Rate ◽  
Basic Hydrolysis ◽  
Rate Profile ◽  
Hydrolysis Of

The hydrolysis of ethyl N-ethylthioncarbamate (ETE) at 100 °C was studied in the range of 7 mol/L HCl to 4 mol/L NaOH. The pH–rate profile showed that the hydrolysis occurred through specific acid catalysis at pH < 2, spontaneous hydrolysis at pH 2–6.5, and specific basic catalysis at pH > 6.5. The Hammett acidity plot and the excess acidity plot against X were linear. The Bunnett–Olsen plot gave a negative slope indicating that the conjugate acid was less hydrated than the neutral substrate. It was concluded that the acid hydrolysis occurred by an A1 mechanism. The neutral species hydrolyzed with general base catalysis shown by the Brønsted plot with β = 0.48 ± 0.04. Water acted as a general base catalyst with (pseudo-)first-order rate constant, kN = 3.06 × 10–7 s–1. At pH > 6.5 the rate constants increased, reaching a plateau at high basicity. The basic hydrolysis rate constant of ethyl N,N-diethylthioncarbamate, which must react by a BAc2 mechanism, increased linearly at 1–3 mol/L NaOH with a second-order rate constant, k2 = 2.3 × 10–4 (mol/L)–1 s–1, which was 10 times slower than that expected for ETE. Experiments of ETE in 0.6 mol/L NaOH with an excess of ethylamine led to the formation of diethyl thiourea, presenting strong evidence that the basic hydrolysis occurred by the E1cb mechanism. In the rate-determining step, the E1cb mechanism involved the elimination of ethoxide ion from the thioncarbamate anion, producing an isothiocyanate intermediate that decomposed rapidly to form ethylamine, ethanol, and COS.Key words: alkylthioncarbamate esters, ethyl N-ethylthioncarbamate, ethyl N,N-diethylthioncarbamate, hydrolysis, mechanism.

scholarly journals Inactivation of the endogenous argininosuccinate lyase activity of duck δ-crystallin by modification of an essential histidine residue with diethyl pyrocarbonate

1993 ◽  
Vol 293 (2) ◽  
pp. 537-544 ◽  
Author(s):  
H J Lee ◽  
S H Chiou ◽  
G G Chang
Keyword(s):  
Enzyme Activity ◽  
Rate Constant ◽  
Order Rate Constant ◽  
Histidine Residue ◽  
Argininosuccinate Lyase ◽  
First Order ◽  
Diethyl Pyrocarbonate ◽  
Order Rate ◽  
Lyase Activity ◽  
Pseudo First Order

The argininosuccinate lyase activity of duck delta-crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and delta-crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of delta-crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the delta-crystallin-L-arginine and delta-crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified delta-crystallin had minimum, if any, conformational changes as compared with the native delta-crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of delta-crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.

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