KINETICS OF HYDROLYSIS OF THE CHROMOGENIC SUBSTRATE S2238 BY ALPHA-THROMBIN : INFLUENCE OF THE pH AND THE IONIC STRENGTH

1987 ◽  
Author(s):  
I M A Verhamme ◽  
G W K van Dedem
Keyword(s):  
Ionic Strength ◽  
Salt Bridge ◽  
Reaction Conditions ◽  
Ionic Strength Dependence ◽  
Strength Dependence ◽  
Activity Decrease ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Optimal Reaction ◽  
Ph Profiles

The knowledge of the pH and ionic strength dependence of kcat and Km for the hydrolysis of S2238 (HD-phe-pip-arg-pNA.2HC1) by alpha-thrombin is essential in determining optimal reaction conditions of residual enzyme in systems where also protease inhibitors and glycosaminoglycan catalysts play a role.We studied the kinetic behavior of S2238 in piperazine/glycylglycine/NaOH buffers from pH 6 to 11 and with a calculated ionic strength up to 0.7 M taking into account the pH-dependent concentration of the buffer species. The kinetic parameters of 60 Michaelis-Menten substrate functions were used for the setup of ionic strength and pH profiles. The kcat values are dependent upon the ionic strength, increasing steeply up to about 0.3 M and decreasing again at high ionic strength.The Km however,reflecting the affinity between enzyme and substrate,is nearly unaffected.The Km values at very alkaline pH are markedly elevated,indicating a conformational form which does not readily bind substrate.The pH profiles for kcat and kcat/Km are displaced towards the low pH side with increasing ionic strength.The ascending limb corresponds to the pK of the Asp-His charge relay system,decreasing with increasing ionic strength from 7.2 to 6.6 in the ES complex and from 6.8 to 6.6 in the free enzyme.Apparently substrate binding provokes a pK increase of the active His residue.The descending limb in the kcat profile could be described by a hypothetical pK varying from 11.5 to 10.7 but the activity decrease is probably due to enzyme inactivation.The alkaline limb of the kcat/Km profile is governed by a pK of 9.4 which is rather independent of the ionic strength and could be attributed to the B-chain terminal isoleucine ,forming a salt bridge with Asp 194 and stabilizing the active site conformation as proven for other serine proteases.Data analysis via a modified Debije function with appropriate estimates for the dielectric constant and the radius of the macro-ion can provide information on the charge density of the enzyme.

Meso tetrakis ortho-, meta-, and para-N-alkylpyridiniopor-phyrins: kinetics of copper(II) and zinc(II) incorporation and zinc porphyrin demetalation

2003 ◽  
Vol 07 (03) ◽  
pp. 139-146 ◽  
Author(s):  
Peter Hambright ◽  
Ines Batinić-Haberle ◽  
Ivan Spasojević
Keyword(s):  
Aqueous Solution ◽  
Ionic Strength ◽  
Effective Charge ◽  
Methyl Ethyl ◽  
Ionic Strength Dependence ◽  
Zinc Porphyrin ◽  
Cationic Porphyrin ◽  
Strength Dependence ◽  
Acid Catalyzed ◽  
Kinetics Of

The relative reactivities of the tetrakis( N -alkylpyridinium- X - yl )-porphyrins where X = 4 (alkyl = methyl, ethyl, n -propyl) , X = 3 (methyl) , and X = 2 (methyl, ethyl, n -propyl, n -butyl, n -hexyl, n -octyl) were studied in aqueous solution. From the ionic strength dependence of the metalation rate constants, the effective charge of a particular cationic porphyrin was usually larger when copper(II) rather than zinc(II) was the reactant. The kinetics of ZnOH + incorporation and the acid catalyzed removal of zinc from the porphyrins in 1.0 M HCl were also studied. In general, the more basic 4- (para-) and 3- (meta-) isomers were the most reactive, followed by the less basic 2- (ortho-) methyl to n -butyl derivatives, with the lipophilic ortho n -hexyl and n -octyl porphyrins the least reactive.

The isomerization of ammonium cyanates. I. The kinetics of ammonium cyanate and alkylammonium cyanate isomerization in dimethyl sulphoxide

1977 ◽  
Vol 30 (4) ◽  
pp. 781 ◽  
Author(s):  
KJ Hall ◽  
DW Watts
Keyword(s):  
Ionic Strength ◽  
Kinetic Studies ◽  
Side Reaction ◽  
Dimethyl Sulphoxide ◽  
Cyanic Acid ◽  
Subsequent Reaction ◽  
Ionic Strength Dependence ◽  
Strength Dependence ◽  
Kinetics Of

The isomerization of ammonium cyanate in dimethyl sulphoxide leads to urea with a substantial side reaction yielding biuret (c. 25 % at 60�). Biuret is formed in a process that is competitive with urea formation and not in a subsequent reaction involving urea. The production of urea, as in water, is by the reaction of ammonia and cyanic acid molecules and it is postulated that biuret is formed through an intermediate of dimerized cyanic acid. Temperature and ionic strength dependence have been studied. Kinetic studies of the isomerization of methylammonium and ethylammonium cyanates are also described.

The ionic strength dependence of chromatin folding

1978 ◽  
Vol 36 (2) ◽  
pp. 220-221
Author(s):  
F. Thoma ◽  
TH. Koller
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Ionic Strength ◽  
Specimen Preparation ◽  
Preparation Technique ◽  
Carbon Supports ◽  
Ionic Strength Dependence ◽  
Chromatin Folding ◽  
Strength Dependence ◽  
Preparation Techniques

Under a variety of electron microscope specimen preparation techniques different forms of chromatin appearance can be distinguished: beads-on-a-string, a 100 Å nucleofilament, a 250 Å fiber and a compact 300 to 500 Å fiber.Using a standardized specimen preparation technique we wanted to find out whether there is any relation between these different forms of chromatin or not. We show that with increasing ionic strength a chromatin fiber consisting of a row of nucleo- somes progressively folds up into a solenoid-like structure with a diameter of about 300 Å.For the preparation of chromatin for electron microscopy the avoidance of stretching artifacts during adsorption to the carbon supports is of utmost importance. The samples are fixed with 0.1% glutaraldehyde at 4°C for at least 12 hrs. The material was usually examined between 24 and 48 hrs after the onset of fixation.

Kinetics of hydrolysis of peroxodisulphate ions in acidic medium to peroxomonosulphuric acid

1981 ◽  
Vol 46 (5) ◽  
pp. 1229-1236 ◽  
Author(s):  
Jan Balej ◽  
Milada Thumová
Keyword(s):  
Ionic Strength ◽  
Rate Constants ◽  
Acidic Medium ◽  
Measured Data ◽  
Molar Ratios ◽  
Starting Solution ◽  
Kinetics Of Hydrolysis ◽  
Rate Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of

The rate of hydrolysis of S2O82- ions in acidic medium to peroxomonosulphuric acid was measured at 20 and 30 °C. The composition of the starting solution corresponded to the anolyte flowing out from an electrolyser for production of this acid or its ammonium salt at various degrees of conversion and starting molar ratios of sulphuric acid to ammonium sulphate. The measured data served to calculate the rate constants at both temperatures on the basis of the earlier proposed mechanism of the hydrolysis, and their dependence on the ionic strength was studied.

scholarly journals A Novel Pseudomonas geniculata AGE Family Epimerase/Isomerase and Its Application in d-Mannose Synthesis

Foods ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1809
Author(s):  
Zhanzhi Liu ◽  
Ying Li ◽  
Jing Wu ◽  
Sheng Chen
Keyword(s):  
3D Structure ◽  
Three Dimensional ◽  
Optimal Temperature ◽  
Reaction Conditions ◽  
Enzymatic Production ◽  
Physiological Properties ◽  
Potential Applications ◽  
Kinetics Of ◽  
Optimal Reaction ◽  
Gene Age

d-mannose has exhibited excellent physiological properties in the food, pharmaceutical, and feed industries. Therefore, emerging attention has been applied to enzymatic production of d-mannose due to its advantage over chemical synthesis. The gene age of N-acetyl-d-glucosamine 2-epimerase family epimerase/isomerase (AGEase) derived from Pseudomonas geniculata was amplified, and the recombinant P. geniculata AGEase was characterized. The optimal temperature and pH of P. geniculata AGEase were 60 °C and 7.5, respectively. The Km, kcat, and kcat/Km of P. geniculata AGEase for d-mannose were 49.2 ± 8.5 mM, 476.3 ± 4.0 s−1, and 9.7 ± 0.5 s−1·mM−1, respectively. The recombinant P. geniculata AGEase was classified into the YihS enzyme subfamily in the AGE enzyme family by analyzing its substrate specificity and active center of the three-dimensional (3D) structure. Further studies on the kinetics of different substrates showed that the P. geniculata AGEase belongs to the d-mannose isomerase of the YihS enzyme. The P. geniculata AGEase catalyzed the synthesis of d-mannose with d-fructose as a substrate, and the conversion rate was as high as 39.3% with the d-mannose yield of 78.6 g·L−1 under optimal reaction conditions of 200 g·L−1d-fructose and 2.5 U·mL−1P. geniculata AGEase. This novel P. geniculata AGEase has potential applications in the industrial production of d-mannose.

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