THROMBIN KINETIC PARAMETERS WITH TRIPEPTIDE p-NITROANALIDES UNDER PHYSIOLOGICALLY RELEVANT CONDITIONS

1987 ◽  
Author(s):  
J W Fenton ◽  
J I Witting ◽  
T M Miller
Keyword(s):  
Active Site ◽  
Active Enzyme ◽  
Chromogenic Substrates ◽  
Human Fibrinogen ◽  
Initial Substrate ◽  
Spontaneous Hydrolysis ◽  
Newton Iterations ◽  
Species Variability ◽  
Do So ◽  
Initial Substrate Concentration

The Michaelis-Menten (Km ), catalytic (kcat ), and specificity (kcat/Km ) constants were determined for humSft α- and β-thrombins with the chromogenic substrate S-2238 (H-D-Phe-Pip-Arg-pNA), Chromozym-TH (Tos-Gly-Pro-Arg-pNA), and Spectrozyme-TH (H-D-HHT-Ala-Arg-pNA) in 0.15 M NaCl buffered with 10 mM HEPES ancTlO mM Tris-HCT at pH 7.4, 37°C. Spontaneous hydrolysis was insignificant for these substrates. Both S-2238 and Spectrozyme-TH exhibited limiting solubilities at ∼35 μM, while Chromozym-TH did not do so up to 50 μM. From initial estimates, Km's and k t's were refined by computer Gause-Newton iterations or nonlinearneast-square fits for the concentration of p-nitroanalide formed per second versus the initial substrate concentration. No major differences were found between a-thrombin (99% α, 91% esterolytically active enzyme, and > 3,500 kilo-U.S. clotting units/g with fibrinogen) and Yγ-thrombin (98% γ. 89% active enzyme, and < 10 kilo-units/g). For α- versus γ-thrombin and the three substrates, respectively, the Km 's were 6.75 ± 0.13 vs 7.62 ± 0.30, 18.4 ± 0.4 vs 23.0 ± 0.3, and 2.53 ± 0.02 vs 3.85 ± 0.51 μM; the kcat> s were 125 ± 1 vs 134 ± 2, 181 ± 2 vs 130 ± 1, and 35.5 ± 0.2 vs 52.4 ± 2.0 s−1 ; and the kcat/Km 's were 18.5 vs 17.6, 9.84 vs 5.65, and 10.1 vs 13.6 s−1 μm . These values closely approximate those of 7.2 ± 0.9 μM, 84 ± 4 s−1 , and 11.7 s−1 μM−1 determined by Higgins, Lewis, and Shafer (J. Biol. Chem. 258:9276-9282, 1983) for the Aα cleavage of human fibrinogen by human α-thrombin under physiologically relevant conditions. Thus, these chromogenic substrates have thrombin specificities similar to that of fibrinogen, although their amidolytic activities are independent of additional active-site regions required for fibrinogen clotting activity (α vs γ-thrombin). Fibrinogen interactions with such active-site regions might account for why the fibrinogen Aa site is an atypical thrombin susceptible bond and the high species variability of fibrinopeptides. (Supported in part by NIH grant HL-13160).

The Physiology of Sea-Urchin Spermatozoa

1950 ◽  
Vol 26 (4) ◽  
pp. 396-409
Author(s):  
LORD ROTHSCHILD
Keyword(s):  
Sea Urchin ◽  
Seminal Plasma ◽  
Human Blood ◽  
Phosphate Buffer ◽  
Dry Weight ◽  
O2 Uptake ◽  
Low Concentrations ◽  
Initial Substrate ◽  
Rough Calculation ◽  
Initial Substrate Concentration

1. Spermatozoa and seminal plasma of Echinus esculentus contain catalase. 2. At 15° C., 4 ml. of a suspension of semen diluted with neutral phosphate buffer in the ratio 1:13 produced in 1 min. 90µl. O2 from an H2O2 solution containing 150 µl. O2. The dry weight of semen in the suspension was 45 mg. and the number of spermatozoa 8.55x109. Under the same conditions, seminal plasma obtained by centrifuging semen produced 50 µl. O2 in 1 min. The dry weight of seminal plasma in the suspension was 12 mg. Human blood, dry weight 229.3 mg./ml., must be diluted with phosphate buffer in the ratio 1:1700 to produce the same amount of O2 in 1 min. as the above suspension of semen. If catalatic activity is defined by the equation Ac = (gt)-1 In {a/(a-x)}, where g = weight in g./ml. of the catalase-containing material, t = 1 min., a = initial substrate concentration (H2O2), and x = amount of H2O2 decomposed in 1 min. at 15° C., Ac = 80-100, 150-200 and 6800 respectively for sea-urchin semen, sea-urchin seminal plasma and human blood. 3. The catalatic activity of semen and seminal plasma is strongly inhibited by hydroxylamine. 4. The O2 uptake and motility of sea-urchin spermatozoa is unaffected by M/5000 H2O2. Higher concentrations of H2O2, M/3000-5000, produce a pronounced ‘shock’ effect, from which the spermatozoa often completely recover. 5. Low concentrations of hydroxylamine, M/3000, reduce O2 uptake and motility. 6. Sea-urchin spermatozoa are almost instantly killed by combinations of hydroxylamine and H2O2, at concentrations which are relatively innocuous when the substances are added separately. 7. A rough calculation indicates that a single spermatozoon contains less than 500 molecules of catalase. 8. A new method of adding H2O2 to catalase-containing material in a manometer is described.

Photodegradation of p-Nitrophenol Catalyzed by ZnO/MWCNTs Composite Catalyst in Water

2012 ◽  
Vol 455-456 ◽  
pp. 1339-1344 ◽  
Author(s):  
Zhe Qi Li ◽  
Jing Yu Liu
Keyword(s):  
Irradiation Time ◽  
Solar Irradiation ◽  
Composite Catalyst ◽  
Catalyst Loading ◽  
Optimal Conditions ◽  
Mass Ratio ◽  
Initial Substrate ◽  
Effects Of Ph ◽  
Potential Use ◽  
Initial Substrate Concentration

Photodegradation ofp-nitrophenol catalyzed by ZnO/MWCNTs composite in water was investigated. The effects of pH, irradiation time, catalyst loading, initial substrate concentration and MWCNTs content on the degradation were investigated. Experiment results revealed that the optimal conditions were ap-nitrophenol concentration of 60.0 mg/L at pH 5.0 with catalyst loading of 10.0 g/L under solar irradiation for the illumination of 180 min. The highest efficiency on photodegradation ofp-nitrophenol can be achieved with an optimal MWCNTs/ZnO mass ratio of 0.16%. Possible decomposing mechanisms were also discussed. The repeatability of photocatalytic activity was tested. The photocatalyst was used ten cycles with degradation efficiency still higher than 95%. The results of the study showed the feasible and potential use of ZnO/MWCNTs composite in degradation of toxic organic pollutants.

Why is the hydrolytic activity of acetylcholinesterase pH dependent? Kinetic study of acetylcholine and acetylthiocholine hydrolysis catalyzed by acetylcholinesterase from electric eel

2018 ◽  
Vol 73 (9-10) ◽  
pp. 345-351 ◽  
Author(s):  
Alena Komersová ◽  
Markéta Kovářová ◽  
Karel Komers ◽  
Václav Lochař ◽  
Alexander Čegan
Keyword(s):  
Catalytic Activity ◽  
Ionic Strength ◽  
Ph Value ◽  
Hydrolytic Activity ◽  
Initial Substrate ◽  
Electric Eel ◽  
Value Range ◽  
Ph Dependent ◽  
Ph Range ◽  
Initial Substrate Concentration

AbstractThe dependence of the activity of acetylcholinesterase from electric eel at a pH value range of 4.8–9.8 (phosphate buffer), regarding acetylcholine and acetylthiocholine hydrolysis, was determined at 25 °C, ionic strength of 0.11 M, and initial substrate concentration of 4 mM. At a pH range of 4.8–9.8, the dependencesA(pH) form a sigmoid increasing curve with the maximum catalytic activity at a pH range 8–9.5. For acetylcholine hydrolysis, the kinetic reason for such an increase inAconsists mainly of an increase in the rate constantk2(Michaelis-Menten) model with increasing pH of the reaction mixture. For acetylthiocholine hydrolysis, the kinetic explication of the determined dependenceA(pH) is more complicated.

Kinetic characterization and inhibition of the rat MAB elastase-2, an angiotensin I-converting serine protease

2002 ◽  
Vol 80 (1) ◽  
pp. 42-47 ◽  
Author(s):  
Carlos F Santos ◽  
Carmem A Paula ◽  
Maria Cristina O. Salgado ◽  
Eduardo Brandt Oliveira
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Peptide Inhibitor ◽  
Ang Ii ◽  
Chromogenic Substrates ◽  
Angiotensin I ◽  
Pancreatic Elastase ◽  
Mesenteric Arterial Bed ◽  
Cleavable Peptide

An elastase-2 has been recently described as the major angiotensin (Ang) II-forming enzyme of the rat mesenteric arterial bed (MAB) perfusate. Here, we have investigated the interaction of affinity-purified rat MAB elastase-2 with some substrates and inhibitors of both pancreatic elastases-2 and Ang II-forming chymases. The Ang II precursor [Pro11-D-Ala12]-Ang I was converted into Ang II by the rat MAB elastase-2 with a catalytic efficiency of 8.6 min–1·µM–1, and the chromogenic substrates N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were hydrolyzed by the enzyme with catalytic efficiencies of 10.6 min–1·µM–1 and 7.6 min–1·µM–1, respectively. The non-cleavable peptide inhibitor CH-5450 inhibited the rat MAB elastase-2 activities toward the substrates Ang I (IC50 = 49 µM) and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (IC50 = 4.8 µM), whereas N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone, an effective active site-directed inhibitor of pancreatic elastase-2, efficiently blocked the Ang II-generating activity of the rat MAB enzyme (IC50 = 4.5 µM). Altogether, the data presented here confirm and extend the enzymological similarities between pancreatic elastase-2 and its rat MAB counterpart. Moreover, the thus far unrealized interaction of elastase-2 with [Pro11-D-Ala12]-Ang I and CH-5450, both regarded as selective for chymases, suggests that evidence for the in vivo formation of Ang II by chymases may have been overestimated in previous investigations of Ang II-forming pathways.Key words: angiotensin, elastase-2, chymase, [Pro11-D-Ala12]-Ang I, CH-5450.

Streptococcus pyogenes Fibronectin-Binding Protein Is a Novel Prothrombin Activator.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1691-1691
Author(s):  
Jonathan I. Creamer ◽  
Peter R. Panizzi ◽  
Paul E. Bock
Keyword(s):  
Fusion Protein ◽  
Streptococcus Pyogenes ◽  
Active Site ◽  
Binding Protein ◽  
Catalytic Site ◽  
Fluorescence Probes ◽  
Binding Studies ◽  
Human Fibrinogen ◽  
Fibronectin Binding Protein ◽  
Fibronectin Binding

Abstract Streptococcus pyogenes fibronectin-binding Protein (SfbX), expressed by emm12 and emm49 strains of S. pyogenes, binds fibronectin through COOH-terminal interactions of the protein.1 SfbX NH2-terminal residues 54–229 share 21% identity to staphylocoagulase (SC) residues 107–321 from Tager 104 strain, which increases to 35% homology when conservative substitutions are considered. SC is a non-proteolytic activator of the central coagulation zymogen prothrombin (ProT). Our structure of the prethrombin 2 complex with a fully active SC fragment (SC(1-325)) showed that SC(1-325) consisted of two α-helical bundle domains and that the NH2-terminal dipeptide is critical for ProT activation via the molecular sexuality mechanism.2 In this mechanism, SC inserts its NH2-terminal Ile1 into the Asp194 pocket of the ProT catalytic domain, inducing conformational activation of the catalytic site. On the basis of its homology, SfbX has been postulated to be a member of the SC family of zymogen activator and adhesion proteins. Plasmids encoding a His6-tagged SfbX(1-312) tobacco etch virus proteinase-cleavable fusion protein and the viral proteinase were co-transformed into E. coli to enable generation of the native SfbX NH2-terminus (Ile-Ser-Asn) during purification. SfbX(1-312) was purified by affinity chromatography on ProT-Affigel and Ni2+-iminodiacetic acid-Sepharose. Active site-specific fluorescent probe labeling of a mixture of ProT and SfbX(1-312) showed covalent labeling of the ProT zymogen, demonstrating that SfbX(1-312) is a non-proteolytic activator of ProT. Incubation of ProT with the NH2-terminally blocked SfbX(1-312) fusion protein did not result in labeling of the active site, indicating that the native NH2-terminus is required for activation, and suggesting that SfbX(1-312) activates ProT through the molecular sexuality mechanism. In ProT activation assays measured by the appearance of chromogenic substrate activity, SfbX(1-312) activated ProT weakly and was more effective in activating prethrombin 1, lacking the fragment 1 domain of ProT. Preliminary binding studies using ProT and thrombin labeled at the catalytic site with fluorescence probes demonstrated binding of SfbX(1-312). SfbX(1-312) decreased thrombin activity toward D-Phe-Pip-Arg-pNA by 75%, with an apparent KD of ~20 nM, indicating that the thrombin catalytic site is perturbed by SfbX binding. Clotting assays showed that neither SfbX(1-312) nor a mixture of SfbX(1-312) and prethrombin 1 clotted human fibrinogen or plasma. Addition of 5 μM SfbX(1-312) to a factor V-dependent plasma clotting assay increased clotting times, indicating that SfbX(1-312) has an inhibitory effect. We conclude that SfbX(1-312) activates ProT conformationally, possibly through the molecular sexuality mechanism. SfbX(1-312) binds tightly to thrombin, perturbing its catalytic site, but does not clot human fibrinogen or plasma. Further studies are needed to determine whether ProT is the pathophysiological target zymogen of SfbX(1-312), to delineate the natural substrate of the SfbX(1-312)·ProT complex, and to elucidate the role of SfbX in the pathology of S. pyogenes infection.

scholarly journals Engineering the properties of a cold active enzyme through rational redesign of the active site

2001 ◽  
Vol 268 (19) ◽  
pp. 5074-5080 ◽  
Author(s):  
Iason Tsigos ◽  
Konstantinos Mavromatis ◽  
Maria Tzanodaskalaki ◽  
Charalambos Pozidis ◽  
Michael Kokkinidis ◽  
...  
Keyword(s):  
Active Site ◽  
Active Enzyme ◽  
Cold Active ◽  
Cold Active Enzyme
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