scholarly journals Kinetic Analysis of Guanidine Hydrochloride Inactivation of β-Galactosidase in the Presence of Galactose

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Charles O. Nwamba ◽  
Ferdinand C. Chilaka
Keyword(s):  
Rate Constants ◽  
Initial Velocity ◽  
Competitive Inhibition ◽  
Guanidine Hydrochloride ◽  
Free Enzyme ◽  
Velocity Data ◽  
Substrate Complex ◽  
Inactivation Rate Constant ◽  
Enzyme Substrate ◽  
Straight Lines

Inactivation of purified β-Galactosidase was done with GdnHCl in the absence and presence of varying [galactose] at 50°C and at pH 4.5. Lineweaver-Burk plots of initial velocity data, in the presence and absence of guanidine hydrochloride (GdnHCl) and galactose, were used to determine the relevant Km and Vmax values, with p-nitrophenyl β-D-galactopyranoside (pNPG) as substrate, S. Plots of ln([P]∞−[P]t) against time in the presence of GdnHCl yielded the inactivation rate constant, A. Plots of A versus [S] at different galactose concentrations were straight lines that became increasingly less steep as the [galactose] increased, showing that A was dependent on [S]. Slopes and intercepts of the 1/[P]∞ versus 1/[S] yielded k+0 and k'+0, the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots of k+0 and k'+0 versus [galactose] showed that galactose protected the free enzyme as well as the enzyme-substrate complex (only at the lowest and highest [galactose]) against GdnHCl inactivation. In the absence of galactose, GdnHCl exhibited some degree of non-competitive inhibition. In the presence of GdnHCl, galactose exhibited competitive inhibition at the lower [galactose] of 5 mM which changed to non-competitive as the [galactose] increased. The implications of our findings are further discussed.

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.

scholarly journals Kinetics of enzymes with iso-mechanisms: analysis of product inhibition

1993 ◽  
Vol 296 (2) ◽  
pp. 355-360 ◽  
Author(s):  
K L Rebholz ◽  
D B Northrop
Keyword(s):  
Rate Constants ◽  
Graphical Representation ◽  
Competitive Inhibition ◽  
Relative Rate ◽  
Additional Term ◽  
Product Inhibition ◽  
Free Enzyme ◽  
Approach Infinity ◽  
Relative Rate Constants ◽  
Kinetic Pattern

Isomerizations of free enzyme can be detected in kinetic patterns of product inhibition when the isomerization is partially rate-limiting. The kinetic pattern is non-competitive, owing to binding of substrate and product to different forms of free enzyme. This adds an additional term to the rate equation, sometimes represented as KSP. Several kineticists have noted that, as the rate of isomerization becomes high in relation to catalytic turnover, the intercept effect will become small, KSP will approach infinity, and the pattern will look competitive. Britton [(1973) Biochem. J. 133, 255-261] asserted that KSP will also approach infinity when the rate of isomerization becomes low. This second assertion is incorrect and can be traced to the particular model and graphical representation used to examine KSP as a function of relative rate constants. The function portrayed as a parabola with two roots for KSP is, instead, a straight line with one root. The algebraic condition justifying the second root obtains in the limit of zero in the rate of reaction and thus is not experimentally relevant, and the appearance of competitive inhibition, based on KSP alone, is not valid. Using a more general model, new equations are derived and presented which provide direct calculations of the apparent rate constants for free enzyme isomerizations from product-inhibition data when the equilibrium of the isomerization is near 1, and useful limits for the rate constants when greater than or less than 1.

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.

scholarly journals Site-directed mutagenesis of β-lactamase I. Single and double mutants of Glu-166 and Lys-73

1990 ◽  
Vol 272 (3) ◽  
pp. 613-619 ◽  
Author(s):  
R M Gibson ◽  
H Christensen ◽  
S G Waley
Keyword(s):  
Rate Constants ◽  
Double Mutant ◽  
Enzyme Mechanism ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Burst Kinetics ◽  
Hydrolysis Of

Two single mutants and the corresponding double mutant of beta-lactamase I from Bacillus cereus 569/H were constructed and their kinetics investigated. The mutants have Lys-73 replaced by arginine (K73R), or Glu-166 replaced by aspartic acid (E166D), or both (K73R + E166D). All four rate constants in the acyl-enzyme mechanism were determined for the E166D mutant by the methods described by Christensen, Martin & Waley [(1990) Biochem. J. 266, 853-861]. Both the rate constants for acylation and deacylation for the hydrolysis of benzylpenicillin were decreased about 2000-fold in this mutant. In the K73R mutant, and in the double mutant, the rate constants for acylation were decreased about 100-fold and 10,000-fold respectively. All three mutants also had lowered values for the rate constants for the formation and dissociation of the non-covalent enzyme-substrate complex. The specificities of the mutants did not differ greatly from those of wild-type beta-lactamase, but the hydrolysis of cephalosporin C by the K73R mutant gave ‘burst’ kinetics.

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

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.

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 inactivation of bovine pancreatic ribonuclease A by bromopyruvic acid

1996 ◽  
Vol 320 (1) ◽  
pp. 187-192 ◽  
Author(s):  
Ming-Hua WANG ◽  
Zhi-Xin WANG ◽  
Kang-Yuan ZHAO
Keyword(s):  
Competitive Inhibition ◽  
Ribonuclease A ◽  
Inactivation Kinetics ◽  
Ionization State ◽  
Substrate Complex ◽  
Irreversible Inhibition ◽  
Enzyme Product ◽  
Enzyme Substrate ◽  
Pancreatic Ribonuclease A ◽  
Kinetics Of

The kinetic theory of substrate reaction during the modification of enzyme activity [Duggleby (1986) J. Theor. Biol. 123, 67–80; Wang and Tsou (1990) J. Theor. Biol. 142, 531–549] has been applied to a study of the inactivation kinetics of ribonuclease A by bromopyruvic acid. The results show that irreversible inhibition belongs to a non-competitive complexing type inhibition. On the basis of the kinetic equation of substrate reaction in the presence of the inhibitor, all microscopic kinetic constants for the free enzyme, the enzyme–substrate complex and the enzyme–product complex have been determined. The non-competitive inhibition type indicates that neither the substrate nor the product affects the binding of bromopyruvic acid to the enzyme and that the ionization state of His-119 may be the same in both the enzyme–substrate and the enzyme–product complexes.

scholarly journals Adenosine 5′-triphosphate sulphurylase from Saccharomyces cerevisiae

1973 ◽  
Vol 133 (3) ◽  
pp. 541-550 ◽  
Author(s):  
Catherine S. Hawes ◽  
D. J. D. Nicholas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Initial Velocity ◽  
Isotopic Exchange ◽  
Competitive Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzyme Reaction ◽  
Velocity Data ◽  
Sequential Reaction ◽  
Optimum Reaction

1. ATP sulphurylase from Saccharomyces cerevisiae was purified 140-fold by using heat treatment, DEAE-cellulose chromatography and Sepharose 6B gel filtration. 2. The enzyme was stable at -15°C, optimum reaction velocity was between pH7.0 and 9.0, and the activation energy was 62kJ/mol (14.7kcal/mol). 3. The substrate was shown to be the MgATP2- complex, free ATP being inhibitory. 4. Double-reciprocal plots from initial-velocity studies were intersecting and the Km of each substrate was determined at infinite concentration of the other (Km MgATP2-, 0.07mm; MoO42-, 0.17mm). 5. Radio-isotopic exchange between the substrate pairs, adenosine 5′-[35S]sulphatophosphate and SO42-, 35SO42- and adenosine 5′-sulphatophosphate, occurred only in the presence of either MgATP2- or PPi. This suggests, along with the initial-velocity data, a sequential reaction mechanism in which both substrates bind before any product is released. 6. The enzyme reaction was specific for ATP and was not inhibited by l-cysteine, l-methionine, SO32-, S2O32- (all 2mm) nor by p-chloromercuribenzoate (1mm). 7. Competitive inhibition of the enzyme with respect to MoO42- was produced by SO42- (Ki=2.0mm) and non-competitive inhibition by sulphide (Ki=3.4mm). 8. Adenosine 5′-sulphatophosphate inhibited strongly and concentrations as low as 0.02mm altered the normal hyperbolic velocity–substrate curves with both MgATP2- and MoO42- to sigmoidal forms.

scholarly journals The Experimentally Determined Velocity of Catalysis could be Higher in the Absence of Sequestration

2019 ◽  
pp. 1-12
Author(s):  
Ikechukwu I. Udema ◽  
Abraham Olalere Onigbinde
Keyword(s):  
Maximum Velocity ◽  
Order Rate Constant ◽  
Free Enzyme ◽  
Theoretical Research ◽  
Coefficient Of Determination ◽  
First Order ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Pseudo First Order ◽  
The Relationship

Background: It is not unusual to observe calculated “total” free enzyme ([E]) in enzyme catalysed reaction, but this should include total enzyme-substrate complex ([EST]) which accounts for sequestration. Objectives: 1) To show indirectly that the velocities of catalytic action can be higher than experimentally observed velocities without sequestration and 2) redefine the relationship between velocity of hydrolysis with Michaelian enzyme and [E], where concentration of substrate, [ST] <  Michaelis-Menten constant, KM. Methods: A theoretical research and experimentation using Bernfeld method to determine velocities of amylolysis with which to mathematically calculate [EST] and the enzyme-substrate complex ([ES]) prepared for product, P, formation. Results: The [EST] is < [E]; [EST] and pseudo-first order constant, k decreased with increasing [ST] and increased with increasing concentration of enzyme [ET] while velocity amylolysis, v and maximum velocity of amylolysis, vmax expectedly increased with increasing [ET] and [ST]. Conclusion: The fact is that the [EST] is lower than what is usually referred to as free enzyme ([ET] - [ES]). Therefore, if the additional part of [EST] dissociated into product within the duration of assay, the velocity of amylolysis could be higher. The most important outcome and corollary when [KM] > [ST] is that v a 1/[E], v a [E][ST] and a quadratic relationship exists between pseudo-first order rate constant and maximum velocity of amylolysis; separately, v is not a [E] and if v a [ST] (if v/[ST] is constant with coefficient of determination = 1), then KM is not applicable.

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