scholarly journals Kinetic analysis of a Michaelis-Menten mechanism in which the enzyme is unstable

1993 ◽  
Vol 294 (2) ◽  
pp. 459-464 ◽  
Author(s):  
C Garrido-del Solo ◽  
F García-Cánovas ◽  
B H Havsteen ◽  
R Varón-Castellanos
Keyword(s):  
Data Analysis ◽  
Experimental Design ◽  
Numerical Simulations ◽  
Kinetic Analysis ◽  
Kinetic Data ◽  
Time Course ◽  
Enzyme Concentration ◽  
Free Enzyme ◽  
Substrate Complex ◽  
Enzyme Substrate

A kinetic analysis of the Michaelis-Menten mechanism is made for the cases in which the free enzyme, or the enzyme-substrate complex, or both, are unstable, either spontaneously or as a result of the addition of a reagent. The explicit time-course equations of all of the species involved has been derived under conditions of limiting enzyme concentration. The validity of these equations has been checked by using numerical simulations. An experimental design and a kinetic data analysis allowing the evaluation of the parameters and kinetic constants are recommended.

scholarly journals Kinetics of an enzyme reaction in which both the enzyme-substrate complex and the product are unstable or only the product is unstable

1994 ◽  
Vol 303 (2) ◽  
pp. 435-440 ◽  
Author(s):  
C Garrido-del Solo ◽  
F García-Cánovas ◽  
B H Havsteen ◽  
E Valero ◽  
R Varón
Keyword(s):  
Computer Simulation ◽  
Data Analysis ◽  
Kinetic Data ◽  
Enzyme Reaction ◽  
Enzyme Concentration ◽  
Substrate Complex ◽  
Use Of Data ◽  
Enzyme Substrate ◽  
Experimental Errors ◽  
Kinetics Of

A kinetic analysis of the Michaelis-Menten mechanism has been made for the case in which both the enzyme-substrate complex and the product are unstable or only the product is unstable, either spontaneously or as the result of the addition of a reagent. This analysis allows the derivation of equations which under conditions of limiting enzyme concentration relate the concentration of all of the species to the time. A kinetic data analysis is suggested, which leads to the evaluation of the kinetic parameters involved in the reaction. The analysis is based on the equation which describes the formation of products with time and one's experimental progress curves. We demonstrate the method numerically by computer simulation of the reaction with added experimental errors and experimentally by the use of data from the kinetic study of the action of tyrosinase on dopamine.

scholarly journals Kinetic analysis of the transient phase and steady state of open multicyclic enzyme cascades.

2005 ◽  
Vol 52 (4) ◽  
pp. 765-780 ◽  
Author(s):  
Ramón Varón ◽  
Bent H Havsteen ◽  
Edelmira Valero ◽  
Milagros Molina-Alarcón ◽  
Francisco García-Cánovas ◽  
...  
Keyword(s):  
Steady State ◽  
Data Analysis ◽  
Kinetic Analysis ◽  
Kinetic Data ◽  
Time Course ◽  
Intrinsic Value ◽  
Transient Phase ◽  
Enzyme Cascade ◽  
Rapid Equilibrium ◽  
Enzyme Cascades

This paper presents a kinetic analysis of the whole reaction course, i.e. of both the transient phase and the steady state, of open multicyclic enzyme cascade systems. Equations for fractional modifications are obtained which are valid for the whole reaction course. The steady state expressions for the fractional modifications were derived from the latter equations since they are not restricted to the condition of rapid equilibrium. Finally, the validity of our results is discussed and tested by numerical integration. Apart from the intrinsic value of knowing the kinetic behaviour of any of the species involved in any open multicyclic enzyme cascade, the kinetic analysis presented here can be the basis of future contributions concerning open multicyclic enzyme cascades which require the knowledge of their time course equations (e.g. evaluation of the time needed to reach the steady state, suggestion of kinetic data analysis, etc.), analogous to those already carried out for open bicyclic cascades.

Transient-Phase and Steady-State Kinetics for Enzyme Activation

1973 ◽  
Vol 51 (6) ◽  
pp. 806-814 ◽  
Author(s):  
Nasrat H. Hijazi ◽  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Kinetic Data ◽  
Enzyme Activation ◽  
Transient Phase ◽  
Time Curve ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Substrate Activation ◽  
Steady State Kinetics ◽  
Special Case

A non-steady-state analysis has been worked out for two mechanisms in which an activator Q can become attached to an enzyme–substrate complex EA, the species EAQ breaking down more rapidly than EA. It is shown that if EAQ breaks down into EQ + product there can be no steady state. If, however, EAQ breaks down into E + Q + product, the transient phase is followed by a steady state in which the product versus time curve is linear. A special case of this mechanism is when Q is the substrate (substrate activation). Some published kinetic data on carboxypeptidase are analyzed with reference to the equations derived.

The influence of pH and inhibitors on the kinetics of enzyme reactions involving two intermediates

1967 ◽  
Vol 45 (5) ◽  
pp. 539-546 ◽  
Author(s):  
Harvey Kaplan ◽  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Ph Dependence ◽  
Free Enzyme ◽  
State Equations ◽  
Enzyme Reactions ◽  
Substrate Complex ◽  
Rate Determining Step ◽  
Enzyme Substrate ◽  
Special Cases ◽  
Influence Of Ph

General steady-state equations are worked out for enzyme reactions which occur according to the scheme [Formula: see text]Equations showing the pH dependence of the kinetic parameters are developed in a form which distinguishes between essential and nonessential ionizing groups. The pK dependence of [Formula: see text], the second-order constant extrapolated to zero substrate constant, gives pK values for groups which ionize on the free enzyme, but reveals such a pK only if the corresponding group is also involved in the breakdown of the Michaelis complex. General steady-state equations are also developed for the case in which an inhibitor can combine with the free enzyme, the enzyme–substrate complex, and also a second intermediate (e.g. an acyl enzyme). The equations are given in a form that is convenient for analyzing the experimental results, and a number of special cases are considered. It is shown how the type of inhibition depends not only on the nature of the inhibitor but also on that of the substrate, an important factor being the rate-determining step of the reaction. Examples of the various kinds of behavior are given.

Total Enzyme-substrate Complex Which Includes Product-destined Enzyme-substrate Complex

2019 ◽  
pp. 1-7
Author(s):  
Ikechukwu I. Udema
Keyword(s):  
Complex Formation ◽  
Maximum Velocity ◽  
Free Enzyme ◽  
Theoretical Research ◽  
Substrate Complex ◽  
Continuous Formation ◽  
Enzyme Substrate ◽  
The Difference ◽  
Total Enzyme

Background: There is no much interest in the determination of total enzyme-substrate complex concentration ([ES]T) which includes undissociated ES that is unaccounted for unlike the usual ES destined for transformation into free enzyme and product or substrate. The reason is speculatively as a result of the lack of awareness of such possibility via sequestration. Objectives: 1) To derive on the basis of both reverse – and standard – quasi-steady – state assumptions equations for the determination of [ES]T which is not restricted to the complex which dissociates to product/substrate and free enzyme and 2) quantitate the value of [ES]T. Methods: A theoretical research and experimentation using Bernfeld method to determine velocities of amylolysis with which to calculate relevant parameters. Results: The [EST] is < [E] ( i. e. [ET] - [ES]); [EST] decreased with increasing [ST] and increased with increasing concentration of enzyme [ET] while the velocity of amylolysis, v and maximum velocity of amylolysis, vmax expectedly increased with increasing [ET] and [ST]. Conclusion: The equations for the determination of the total enzyme-substrate complex, free enzyme without any complex formation before and after dissociation of enzyme-complex into product and/or substrate and free enzyme were derived. The difference, [ET] - [ES] is a heterogeneous mixture of undissociated ES and free enzyme without any complex formation. This is the case because [ES] which dissociates into product is only a part of the total enzyme-substrate complex. There is a continuous formation of ES during and at the expiry of the duration of assay as long as there is no total substrate depletion.

Mechanism of Collagen: Glucosyl-transferase and its Relation to Platelet: Collagen Adhesion

1975 ◽  
Author(s):  
D. F. Smith ◽  
D. P. Kosow ◽  
G. A. Jamieson
Keyword(s):  
Steady State ◽  
Cell Surface ◽  
Platelet Function ◽  
Kinetic Data ◽  
Soluble Form ◽  
Physiological Conditions ◽  
Substrate Complex ◽  
Enzymatic Mechanism ◽  
Enzyme Substrate ◽  
Induced Aggregation

Elucidation of the enzymatic mechanism of collagen: glucosyltransferase is essential to an understanding of its role in platelet function. A soluble form of the enzyme has been purified 100-fold and a sensitive new assay system developed. Studies with effectors such as UDP, ADP and ristocetin under steady state conditions have shown that only two of the possible sequential mechanisms are consistent with the kinetic data. Inhibition by UDP and ADP is competitive with UDPG but non-competitive with galactosylhydroxylysine. They would not, therefore, be expected to inhibit the formation of an enzyme-substrate complex with collagen. Under physiological conditions, their presence would be expected to increase the affinity of the cell surface enzyme for its acceptor on collagen in the case of the ordered mechanism, or not to affect it in the case of the random mechanism. These data are consistent with the potentiation of collagen-induced aggregation by ADP, and the lack of effect of UDP on the adherence of platelets to collagen.(Supported, in part, by USPHS.)

Final Phase of Enzyme Reactions Following a Michaelis-Menten Mechanism in which the Free Enzyme and/or the Enzyme-Substrate Complex are Unstable

1994 ◽  
Vol 375 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Ramón Varón ◽  
Carmelo Garrido del Solo ◽  
Manuela Garcίa-Moreno ◽  
Angela Sánchez-Gracia ◽  
Francisco Garcίa-Cánovas
Keyword(s):  
Free Enzyme ◽  
Final Phase ◽  
Enzyme Reactions ◽  
Substrate Complex ◽  
Enzyme Substrate

scholarly journals Evaluation of the extent of the binding site in human tissue kallikrein by synthetic substrates with sequences of human kininogen fragments

1995 ◽  
Vol 312 (1) ◽  
pp. 233-238 ◽  
Author(s):  
E Del Nery ◽  
J R Chagas ◽  
M A Juliano ◽  
E S Prado ◽  
L Juliano
Keyword(s):  
Human Tissue ◽  
Kinetic Data ◽  
Time Course ◽  
Tissue Kallikrein ◽  
Hydrolysis Time ◽  
Porcine Tissue ◽  
Enzyme Substrate ◽  
Substrate Side ◽  
Kinetics Of ◽  
Hydrolysis Of

We have synthesized internally quenched peptides spanning the Met379-Lys380 or Arg389-Ser390 bonds of human kininogen (hkng) that flank lysyl-bradykinin and have studied the kinetics of their hydrolysis by human tissue kallikrein. The kinetic data for the hydrolysis of the Met-Lys bond in substrates with an N-terminal extension showed that interactions up to position residue P10 contribute to the efficiency of cleavage. In contrast, there were no significant variations in the kinetic data for the hydrolysis of substrates with C-terminal extensions at sites P′4 to P′11. A similar pattern was observed for the cleavage of substrates containing an Arg-Ser bond because substrates extended up to residue P6 were hydrolysed with the highest kcat/Km values in the series, whereas those extended to P′11 on the C-terminal side had a lower susceptibility to hydrolysis. Time-course studies of hydrolysis by human and porcine tissue kallikreins of a Leu373 to Ile393 human kininogen fragment containing omicron-aminobenzoic acid (Abz) at the N-terminus and an amidated C-terminal carboxyl group Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg- Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Ile-NH2 (Abz-[Leu373-Ile393]-hkng-NH2) indicated that the cleavage of Met-Lys and Arg-Ser bonds in the same molecule occurs via the formation of independent enzyme-substrate complexes. The hydrolysis of Abz-F-R-S-S-R-Q-EDDnp [where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] and Abz-M-I-S-L-M-K-R-P-Q-EDDnp by human tissue kallikrein had maximal kcat/Km values at pH 9-9.5 for both substrates. The pH-dependent variations in this kinetic parameter were almost exclusively due to variations in kcat. A significant decrease in kcat/Km values was observed for the hydrolysis of Arg-Ser and Met-Lys bonds in the presence of 0.1 M NaCl. Because this effect was closely related to an increase in Km, it is likely that sodium competes with the positive charges of the substrate side chains for the same enzyme subsites.

scholarly journals Kinetic behaviour of zymogen activation processes in the presence of an inhibitor

1993 ◽  
Vol 290 (2) ◽  
pp. 463-470 ◽  
Author(s):  
R Varón ◽  
M C Manjabacas ◽  
M García-Moreno ◽  
E Valero ◽  
F Garcia-Canovas
Keyword(s):  
Data Analysis ◽  
Kinetic Analysis ◽  
General Model ◽  
Kinetic Data ◽  
Kinetic Equations ◽  
Zymogen Activation ◽  
Activation Model ◽  
Kinetic Behaviour ◽  
Irreversible Inhibition

A global kinetic analysis of a general zymogen activation model, where not only the activating but also the activated enzyme suffer an irreversible inhibition is presented. A reaction in which the enzyme acts upon a substrate is coupled to monitor the process. In addition, we determined the corresponding kinetic equations for a number of particular cases of the general model studied. Finally, a kinetic data analysis and a procedure to discriminate among the different mechanisms considered, which are based on the kinetic equations obtained, are suggested.

BTX Degradation: The concept of microbial integration

2019 ◽  
Author(s):  
Chem Int
Keyword(s):  
Gas Chromatography ◽  
Soil Sample ◽  
High Activity ◽  
Niger Delta ◽  
Batch Reactor ◽  
Free Enzyme ◽  
Delta Area ◽  
Substrate Complex ◽  
Fungal Activity ◽  
Enzyme Substrate

The concept of microbial integration was carried out to examine bacterial and fungal activity on bezene, toluence and xylene (BTX) degradation in a batch reactor. The investigation was conducted for thirty five day of exposure of contact of members and substrate which yielded enzyme substrate complex as well disintegrated to produce products and free enzyme. Bacterial and fungal concentration was monitored per week and the results obtained recorded. The gas chromatography results of Ngara soil sample investigated reveals the concentration of M, P, and O – Xylene for different days of exposure. Increase in both bacterial and fungal was experienced with decrease in BTX concentration, whereas increase in bacterial is more than fungi, indicating the high activity of bacterial in the reactor than that of fungi. Although, both were well integrated in bioremediation program to enhance the effective remediation of BTX contaminants in Ngara soil, Omuigwe Alun Community, Niger Delta Area of Nigeria.

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