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.

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 Single-turnover and steady-state kinetics of hydrolysis of cephalosporins by β-lactamase I from Bacillus cereus

1985 ◽  
Vol 231 (1) ◽  
pp. 83-88 ◽  
Author(s):  
R Bicknell ◽  
S G Waley
Keyword(s):  
Steady State ◽  
Bacillus Cereus ◽  
Substrate Complex ◽  
Single Turnover ◽  
Rate Determining Step ◽  
Enzyme Substrate ◽  
Steady State Kinetics ◽  
Lactam Ring ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of the hydrolysis of two cephalosporins by β-lactamase I from Bacillus cereus 569/H/9 has been studied by single-turnover and steady-state methods. Single-turnover kinetics could be measured over the time scale of minutes when cephalosporin C was the substrate. The other substrate, 7-(2′,4′-dinitrophenylamino)deacetoxycephalosporanic acid, was hydrolysed even more slowly, and has potential for use in crystallographic studies of β-lactamases. Comparison of single-turnover and steady-state kinetics showed that, for both substrates, opening the β-lactam ring (i.e. acylation of the enzyme) was the rate-determining step. Thus the non-covalent enzyme-substrate complex is expected to be the intermediate observed crystallographically.

pH-dependence of the fast step of maltose hydrolysis catalysed by glucoamylase G1 from Aspergillus niger

2000 ◽  
Vol 349 (2) ◽  
pp. 623-628 ◽  
Author(s):  
Ulla CHRISTENSEN
Keyword(s):  
Aspergillus Niger ◽  
Ph Dependence ◽  
Acid Group ◽  
Reaction Scheme ◽  
Free Enzyme ◽  
Pka Values ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Ph Range ◽  
State Kinetic

The presteady-state kinetic parameters of the interaction of wild-type glucoamylase from Aspergillus niger (EC 3.2.1.3) with maltose were obtained and analysed in the pH range 3-7 with intervals of 0.25 pH units. In all cases the following three-step reaction scheme was found to apply. E+S ES1 ES2 E+P The general result of the analysis of the presteady-state kinetics is that glucoamylase G1 is affected by the protonation states of three groups, with pKa values of 2.7, 4.5 and 5.7 in the free enzyme and of 2.7, 4.75 and 6.5 in the first enzyme-substrate complex. The protonation of the group in the enzyme-substrate complex with a pKa 6.5 had no effect on k2 (1640 s-1) or k-2 (20±4 s-1), but resulted in a stronger enzyme-substrate interaction, due to a decrease of K1 from 40 to 6.3 mM. In other words, when the substrate is bound, the pKa of the acid group changes to increase the fraction of reactive enzyme. Since this pKa parallels that of the Michaelis complex, known from the pH-dependence of kcat, the group in question is most probably the catalytic acid Glu-179. Protonation of Glu-179 thus is of no importance in the second step, clearly indicating that this step represents a conformational change and not the actual hydrolysis step of the reaction. Protonation of the pKa = 4.75 group leads to a small decrease in k2 to 1090 s-1, and also to minor changes in K1. The group with pKa = 2.7 leads to a major decrease of k2, of which the limit may be zero, but shows no effect on K1. Thus no difference is seen between the pKa values of the free enzyme and of the first enzyme-substrate complex at low pH.

Transient-phase studies of a trypsin-catalyzed reaction

1970 ◽  
Vol 48 (12) ◽  
pp. 1793-1802 ◽  
Author(s):  
H. P. Kasserra ◽  
K. J. Laidler
Keyword(s):  
Steady State ◽  
Ph Dependence ◽  
Enzyme Concentration ◽  
Alcohol Concentration ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Steady State Kinetics ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester

The stopped-flow technique has been used to study the pre-steady-state kinetics of the hydrolysis of N-carbobenzoxy-L-alanine-p-nitrophenyl ester catalyzed by trypsin. By working under conditions such that the enzyme concentration is much greater than that of the substrate, it has been possible to measure [Formula: see text] the rate constant for the conversion of the enzyme-substrate complex into the acyl enzyme. The pH dependence of [Formula: see text] reveals a pKb′ value of 6.9 for the conversion of complex into acyl enzyme, in agreement with deductions from steady-state investigations. The pH dependence of [Formula: see text] (equal to k−1 + k2)/k1) has also been determined. The results provide direct evidence for the existence of an enzyme-substrate complex for this reaction.The work has been done in various mixtures of water and isopropyl alcohol. The logarithms of the rate constants [Formula: see text] and [Formula: see text] vary linearly with 1/D, showing a decrease with increasing alcohol concentration; [Formula: see text] increases with alcohol concentration. The solvent results suggest that addition of alcohol affects the hydrophobic bonding in the protein and leads to unfolding of the enzyme.

scholarly journals The kinetics of the reaction of nitrophenyl phosphates with alkaline phosphatase from Escherichia coli

1968 ◽  
Vol 106 (2) ◽  
pp. 455-460 ◽  
Author(s):  
D. R. Trentham ◽  
H. Gutfreund
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Ph Dependence ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Nitrophenyl Phosphate ◽  
Steady State Rate ◽  
State Rate ◽  
Rate Of Hydrolysis ◽  
Ph Range

1. The steady-state rate of hydrolysis of 2,4-dinitrophenyl phosphate catalysed by Escherichia coli phosphatase is identical with that of 4-nitrophenyl phosphate over the pH range 5·5–8·5. 2. The increase in the rate of the enzyme-catalysed decomposition of nitrophenyl phosphates in the presence of tris at pH8·1 and 5·9 is consistent with the hypothesis that tris increases the rate of decomposition of a phosphoryl-enzyme intermediate. At pH8·1 the rate of decomposition of the phosphoryl-enzyme is approximately twice as fast as the rate of its formation, whereas at pH5·9 the rate of formation of the phosphoryl-enzyme is considerably faster than its decomposition. 3. Pre-steady-state measurements of the initial transient of the liberation of 2,4-dinitrophenol during the reaction of the enzyme with 2,4-dinitrophenyl phosphate confirmed the above pH-dependence of the ratio of the rates of phosphorylation and dephosphorylation of the enzyme. At optimum pH (above pH8), when the phosphorylation of the enzyme by the substrate is rate-determining, this step must be controlled by a rearrangement of the enzyme or enzyme–substrate complex.

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.

KINETIC EQUATIONS FOR ENZYME REACTIONS IN THE PRESENCE OF POWERFUL INHIBITORS

1959 ◽  
Vol 37 (8) ◽  
pp. 1268-1271 ◽  
Author(s):  
Richard M. Krupka ◽  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Kinetic Equations ◽  
Competitive Inhibitor ◽  
Graphical Analysis ◽  
Rate Data ◽  
State Equations ◽  
Enzyme Reactions ◽  
Simple Addition

Steady-state equations are worked out for the case of a competitive inhibitor that is present in concentrations comparable with that of the enzyme; allowance is made for the inhibitor attached to the enzyme. Two cases are considered: in case 1 the enzyme and inhibitor form a simple addition complex, while in case 2 a molecule is split off. Methods of graphical analysis of rate data are described.

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.)

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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