Kinetic studies of prothrombin activation: effect of factor Va and phospholipids on formation of the enzyme-substrate complex

Biochemistry ◽  
1984 ◽  
Vol 23 (20) ◽  
pp. 4557-4564 ◽  
Author(s):  
Jan L. M. L. Van Rijn ◽  
Jose W. P. Govers-Riemslag ◽  
Robert F. A. Zwaal ◽  
Jan Rosing
Keyword(s):  
Kinetic Studies ◽  
Activation Effect ◽  
Substrate Complex ◽  
Factor Va ◽  
Enzyme Substrate ◽  
Prothrombin Activation

scholarly journals Allosteric Enzyme-Based Biosensors—Kinetic Behaviours of Immobilised L-Lysine-α-Oxidase from Trichoderma viride: pH Influence and Allosteric Properties

Biosensors ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 145
Author(s):  
Antonio Guerrieri ◽  
Rosanna Ciriello ◽  
Giuliana Bianco ◽  
Francesca De Gennaro ◽  
Silvio Frascaro
Keyword(s):  
Trichoderma Viride ◽  
Kinetic Studies ◽  
Pt Electrode ◽  
Allosteric Enzyme ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Ph Influence ◽  
Ph Dependent ◽  
Kinetics Of ◽  
Allosteric Properties

The present study describes the kinetics of L-lysine-α-oxidase (LO) from Trichoderma viride immobilised by co-crosslinking onto the surface of a Pt electrode. The resulting amperometric biosensor was able to analyse L-lysine, thus permitting a simple but thorough study of the kinetics of the immobilised enzyme. The kinetic study evidenced that LO behaves in an allosteric fashion and that cooperativity is strongly pH-dependent. Not less important, experimental evidence shows that cooperativity is also dependent on substrate concentration at high pH and behaves as predicted by the Monod-Wyman-Changeux model for allosteric enzymes. According to this model, the existence of two different conformational states of the enzyme was postulated, which differ in Lys species landing on LO to form the enzyme–substrate complex. Considerations about the influence of the peculiar LO kinetics on biosensor operations and extracorporeal reactor devices will be discussed as well. Not less important, the present study also shows the effectiveness of using immobilised enzymes and amperometric biosensors not only for substrate analysis, but also as a convenient tool for enzyme kinetic studies.

Synthesis and Kinase Phosphorylation of 4-Deoxy-D-threohexulose

1973 ◽  
Vol 51 (7) ◽  
pp. 969-972 ◽  
Author(s):  
Clifford Raymond Haylock ◽  
Keith Norman Slessor
Keyword(s):  
Lithium Aluminum Hydride ◽  
Aluminum Hydride ◽  
Kinetic Studies ◽  
Lithium Aluminum ◽  
Substrate Complex ◽  
Acetobacter Suboxydans ◽  
Enzyme Substrate ◽  
Kinase Phosphorylation ◽  
Hydrolysis Of ◽  
Yeast Hexokinase

Synthesis of the only unknown deoxyfructose, 4-deoxy-D-threohexulose, is reported. Its preparation involved reductive lithium aluminum hydride ring opening of 3,4-anhydro-1,2:5,6-di-O-isopropylidene- D-talitol, followed by hydrolysis of the resulting epimeric deoxy diisopropylidene hexitols and selective Acetobacter suboxydans oxidation of 3-deoxy-D-arabinohexitol. Kinetic studies using 4-deoxy-D-threohexulose as substrate for yeast hexokinase support the premise that the C-4 hydroxyl is a binding group in formation of the enzyme–substrate complex. Enzymatic synthesis of 4-deoxy-D-threohexulose 6-phosphate and 4-deoxy-D-threohexulose 1,6-diphosphate has been achieved in low yield from 4-deoxy-D-threohexulose.

Kinetic Studies of the Flagellar Movement of Sea-Urchin Spermatozoa

1969 ◽  
Vol 50 (1) ◽  
pp. 203-222
Author(s):  
M. E. J. HOLWILL
Keyword(s):  
Dielectric Constant ◽  
Ionic Strength ◽  
Sea Urchin ◽  
Activation Parameters ◽  
Kinetic Studies ◽  
Basic Part ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Glycerinated Model ◽  
Rate Limiting

1. The movements of intact and glycerinated spermatozoa from two species of sea-urchin were examined in the range of temperature between 5 and 30° C. The variations of the frequency of the glycerinated spermatozoa with changing pH, dielectric constant and ionic strength were examined. 2. From the temperature studies values for activation entropies and enthalpies were obtained that pertain to the chemical reaction which limits the frequency. 3. Within the limits of experimental error the activation parameters are identical for the intact spermatozoa and for the glycerinated model of the same species. The results are consistent with the hypothesis that the rate-limiting reaction is the breakdown of an enzyme-substrate complex. 4. The pH studies suggest that neither the acidic nor the basic part of the enzyme is involved in complex formation but that both participate in the breakdown of the complex. 5. The results obtained from the studies of dielectric constant are interpreted in terms of a model for the breakdown of the enzyme-substrate complex involving the separation of several spherical charges. 6. The studies of pH and ionic strength suggest that both 14 S and 30 S dynein participate in the mechano-chemical process responsible for bending the flagellum.

scholarly journals Paenibacillus sp. TS12 glucosylceramidase: kinetic studies of a novel sub-family of family 3 glycosidases and identification of the catalytic residues

2004 ◽  
Vol 378 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Krisztina PAAL ◽  
Makoto ITO ◽  
Stephen G. WITHERS
Keyword(s):  
Kinetic Studies ◽  
Acid Base ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
General Acid ◽  
Dependent Inactivation ◽  
Paenibacillus Sp ◽  
Continuous Assay ◽  
Reduced Activity ◽  
Enzyme Intermediate

GCase (glucosylceramidase) from Paenibacillus sp. TS12, a family 3 glycosidase, hydrolyses the β-glycosidic linkage of glucosylceramide with retention of anomeric configuration via a two-step, double-displacement mechanism. Two carboxyl residues are essential for catalysis, one functioning as a nucleophile and the other as a general acid/base catalyst. p-Nitrophenyl β-d-glucopyranoside [Km=0.27±0.02 mM and kcat/Km=(2.1±0.2)×106 M−1·s−1] and 2,4-dinitrophenyl β-d-glucopyranoside [Km=0.16±0.02 mM and kcat/Km=(2.9±0.4)×106 M−1·s−1] were used for continuous assay of the enzyme. The dependence of kcat (and kcat/Km) on pH revealed a dependence on a group of pKa≤7.8 in the enzyme–substrate complex which must be protonated for catalysis. Incubation of GCase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-d-glucopyranoside caused time-dependent inactivation (Ki=2.4±0.7 mM and ki=0.59±0.05 min−1) due to the accumulation of a trapped glycosyl–enzyme intermediate. Electrospray ionization MS analysis of the peptic digest of this complex showed that the enzyme was covalently labelled by the reagent at Asp-223, consistent with its role as nucleophile. A mutant modified at this residue (D223G) showed substantially reduced activity compared with the wild type (>104), but this activity could be partially restored by addition of formate as an external nucleophile. Kinetic analysis of the mutant E411A indicated that Glu-411 serves as the general acid/base catalytic residue since this mutant was pH-independent and since considerable GCase activity was restored upon addition of azide to E411A, along with formation of a glycosyl azide product.

scholarly journals Kinetic Studies on the Enzyme-substrate Complex Formation of p-Hydroxybenzoate Hydroxylase by the Stopped-flow Method

1972 ◽  
Vol 27 (10) ◽  
pp. 1172-1175 ◽  
Author(s):  
Naoki Higashi ◽  
Hirohumi Shoun ◽  
Keiji Yano ◽  
Κει Arima ◽  
Keitaro Hiromi
Keyword(s):  
Complex Formation ◽  
Reduction Rate ◽  
Kinetic Studies ◽  
Stopped Flow ◽  
Apparent Velocity ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Hydroxybenzoate Hydroxylase ◽  
Anaerobic Reduction

The spectrophotometric and spectrofluorometric investigations of the enzyme-substrate complex formation of p-hydroxybenzoate hydroxylase was made by the stopped-flow technique. The apparent velocity of the formation of the enzyme-substrate complex (the velocity of the absorbance change in visible and UV regions, and the velocity of the quenching of the fluorescence intensity in the FAD moiety of the holoenzyme by the substrate) was rapid enough to explain the maximal overall velocity (72 sec-1) or the activated anaerobic reduction rate (kredmax= 200 sec-1). The results were consistent with a two-step mechanism involving a rapid bimolecular association of enzyme and substrate, and a slower follow-up unimolecular process.

scholarly journals Photoenzymatic Repair of Ultraviolet Damage in DNA

1962 ◽  
Vol 45 (4) ◽  
pp. 725-741 ◽  
Author(s):  
Claud S. Rupert
Keyword(s):  
Heavy Metals ◽  
Quantum Yield ◽  
Kinetic Studies ◽  
Order Rate Constant ◽  
Reaction Scheme ◽  
First Order ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Exponential Decrease ◽  
Ultraviolet Damage

The photoenzyme from bakers' yeast which repairs ultraviolet-inactivated transforming DNA is mechanically bound to ultraviolet-irradiated DNA in the dark, but not to unirradiated DNA. In the bound condition it is stabilized against inactivation by heat and heavy metals. Both the mechanical binding and stabilization are eliminated by illumination. These observations are consistent with the reaction scheme suggested by kinetic studies, in which the enzyme combines with the ultraviolet lesions in DNA and the complex absorbs light, producing repair and subsequent liberation of the enzyme. The approximately exponential decrease of heat stabilization during illumination gives the first order rate constant for the light-dependent step at the corresponding light intensity. This quantity in turn sets limits on the possible magnitude of the molar absorption coefficient of the enzyme-substrate complex and on the quantum yield of the process.

scholarly journals Photoenzymatic Repair of Ultraviolet Damage in DNA

1962 ◽  
Vol 45 (4) ◽  
pp. 703-724 ◽  
Author(s):  
Claud S. Rupert
Keyword(s):  
Dna Repair ◽  
Competitive Inhibition ◽  
Kinetic Studies ◽  
Reaction Scheme ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Light Intensities ◽  
Presence And Absence ◽  
Ultraviolet Damage ◽  
Kinetics Of

As previously reported, ultraviolet-inactivated bacterial transforming DNA can be restored to activity by an enzyme-like agent from bakers' yeast which requires light for its activity. Kinetics of this reaction, in the presence and absence of inhibitors, are found consistent with the Michaelis-Menten reaction scheme, with the sites of ultraviolet damage on the DNA serving as substrate and the repaired structure as product. Kinetic studies with different light intensities suggest that the necessary illumination causes photolysis of the enzyme-substrate complex with concurrent repair of the DNA. Competitive inhibition of irradiated transforming DNA repair, which occurs when irradiated non-transforming DNA is present in the same reaction mixture, permits ultraviolet damage (of the kind capable of being photoreactivated) to be detected in any type of DNA.

scholarly journals Kinetic studies of the polygalacturonase enzyme from Colletotrichum lindemuthianum

1992 ◽  
Vol 285 (2) ◽  
pp. 551-556 ◽  
Author(s):  
G Waksman ◽  
G Turner ◽  
A R Walmsley
Keyword(s):  
Steady State ◽  
Fluorescence Intensity ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Colletotrichum Lindemuthianum ◽  
Protein Fluorescence ◽  
Substrate Complex ◽  
Single Turnover ◽  
Enzyme Substrate ◽  
Rapid Quench

The intrinsic protein fluorescence of the polygalacturonase from Colletotrichium lindemuthianum was exploited in stopped-flow experiments aimed at elucidating the kinetic mechanism for this enzyme. Binding of the polymeric substrate polygalacturonic acid (PGA) essentially produced a triphasic fluorescence profile. There was an initial rapid quench in fluorescence, consistent with the rapid formation of the enzyme-substrate complex, with an equilibrium constant of about 8 x 10(-4)% (w/v) PGA (about 0.27 microM). There then followed a near-constant fluorescence phase, attributable to turnover of the enzyme-substrate complex as a steady-state intermediate. As the concentration of the steady-state intermediate became depleted, towards the end of the reaction, there was a partial return of the fluorescence intensity. This phase is attributed to a final, single turnover of the enzyme at the end of the reaction. The fluorescence intensity does not return to its original level due to product remaining bound at the end of the reaction.

The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

1980 ◽  
Vol 45 (2) ◽  
pp. 427-434 ◽  
Author(s):  
Kveta Heinrichová ◽  
Rudolf Kohn
Keyword(s):  
Acetyl Derivative ◽  
Competitive Inhibitor ◽  
Hydroxyl Groups ◽  
Pectic Acid ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Individual ◽  
Degradation Degree ◽  
Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

scholarly journals Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli

2021 ◽  
Vol 14 ◽  
pp. 117863612110246
Author(s):  
Cheuk Yin Lai ◽  
Ka Lun Ng ◽  
Hao Wang ◽  
Chui Chi Lam ◽  
Wan Keung Raymond Wong
Keyword(s):  
Escherichia Coli ◽  
Cellulolytic Bacterium ◽  
Systematic Screening ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Glycosylation Sites ◽  
Endogenous Proteins ◽  
Cellulose Binding

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as ‘attributive tags’ for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

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