scholarly journals A graphical method for determining inhibition parameters for partial and complete inhibitors

1987 ◽  
Vol 248 (3) ◽  
pp. 815-820 ◽  
Author(s):  
M Yoshino
Keyword(s):  
Substrate Concentration ◽  
Competitive Inhibition ◽  
Graphical Method ◽  
Enzyme Reaction ◽  
Reaction Rate Constant ◽  
Inhibition Mechanism ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Characteristic Features ◽  
Substrate Inhibitor

A new simple graphical method is described for the determination of inhibition type and kinetic parameters of an enzyme reaction without any replot. The method consists of plotting experimental data as v/(vo-v) versus the reciprocal of the inhibitor concentration at different substrate concentrations, where v and vo represent the velocity in the presence and in the absence of the inhibitor respectively with a given concentration of the substrate. Partial inhibition gives straight lines that converge on the abscissa at a point away from the origin, whereas complete inhibition gives lines that go through the origin. The inhibition constants of enzymes and the reaction rate constant of the enzyme-substrate-inhibitor complex can be calculated from the abscissa and ordinate intercepts of the plot. The relationship between the slope of the plot and the substrate concentration shows characteristic features depending on the inhibition type: for partial competitive inhibition, the straight line converging on the abscissa at-Ks, the dissociation constant of the enzyme-substrate complex; for non-competitive inhibition, a constant slope independent of the substrate concentration; for uncompetitive inhibition, a hyperbola decreasing with the increase in the substrate concentration; for mixed-type inhibition, a hyperbola increasing with the increase in the substrate concentration. The properties of the replot are useful in confirmation of the inhibition mechanism.

Functional analysis of hyperthermophilic endocellulase from Pyrococcus horikoshii by crystallographic snapshots

2011 ◽  
Vol 437 (2) ◽  
pp. 223-230 ◽  
Author(s):  
Han-Woo Kim ◽  
Kazuhiko Ishikawa
Keyword(s):  
Enzyme Reaction ◽  
Intermediate Structure ◽  
Amino Acid Residues ◽  
Pyrococcus Horikoshii ◽  
Substrate Complex ◽  
Substrate Structure ◽  
Enzyme Substrate ◽  
Cellulose Substrates ◽  
Boat Form ◽  
Low Activity

A hyperthermophilic membrane-related β-1,4-endoglucanase (family 5, cellulase) of the archaeon Pyrococcus horikoshii was found to be capable of hydrolysing cellulose at high temperatures. The hyperthermophilic cellulase has promise for applications in biomass utilization. To clarify its detailed function, we determined the crystal structures of mutants of the enzyme in complex with either the substrate or product ligands. We were able to resolve different kinds of complex structures at 1.65–2.01 Å (1 Å=0.1 nm). The structural analysis of various mutant enzymes yielded a sequence of crystallographic snapshots, which could be used to explain the catalytic process of the enzyme. The substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite −1, and the β-1,4-glucoside bond between glucose moieties is twisted between subsites −1 and +1. Subsite −2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. This type of recognition is important for creation of the distorted boat form of the substrate at subsite −1. A rare enzyme–substrate complex was observed within the low-activity mutant Y299F, which suggested the existence of a trapped ligand structure before the formation by covalent bonding of the proposed intermediate structure. Analysis of the enzyme–substrate structure suggested that an incoming water molecule, essential for hydrolysis during the retention process, might be introduced to the cleavage position after the cellobiose product at subsites +1 and +2 was released from the active site.

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 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 Factors like Dilution and Mixing Influence Enzymatic Reactions

2020 ◽  
Vol 19 (4) ◽  
Author(s):  
Mahin Basha Syed ◽  
Venkatanagraraju Erumalla
Keyword(s):  
Enzyme Activity ◽  
Substrate Concentration ◽  
Enzymatic Reaction ◽  
Enzyme Reaction ◽  
Enzyme Concentration ◽  
Enzymatic Reactions ◽  
First Report ◽  
Enzyme Substrate ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

Enzyme-catalyzed reactions were influenced by many factors. The enzyme reacts with the substrate and converts it into products. Enzymes are influenced by temperature, pH, enzyme concentration, and substrate concentration. This paper evaluates the hypothesis of factors that may influence enzyme activity. Two more factors that affects enzyme activity are dilution and mixing. In enzyme-substrate reactions, the small amount of dilution and mixing will not affect the enzyme activity. Dilution and mixing do not slowdowns the enzyme reaction but it enhances the enzymatic reaction up to a certain limit. Increase in dilution results in less interaction of enzyme substrate, which causes a decrease in the rate of reactions. To the best of our knowledge, this is the first report to shows that, factors like mixing and dilution also affect enzyme and substrate reactions.

scholarly journals The pre-eminence of kcat. in the manifestation of optimal enzymic activity delineated by using the Briggs-Haldane two-step irreversible kinetic model

1976 ◽  
Vol 159 (1) ◽  
pp. 165-166 ◽  
Author(s):  
K Brocklehurst ◽  
A Cornish-Bowden
Keyword(s):  
Kinetic Model ◽  
Dissociation Constant ◽  
Substrate Concentration ◽  
Product Formation ◽  
Enzymic Activity ◽  
Substrate Complex ◽  
Diffusion Limited ◽  
Enzyme Substrate

The suggestion by Fersht [(1974) Proc. R. Soc. London Ser. B 187, 397-407] that enzymes that provide maximal rates of catalysis should be characterized by values of Ks, the dissociation constant of the enzyme-substrate complex, greater than 10 times the value of the ambient substrate concentration has been examined. 2. For such enzymes, Ks is not relevant, and attention is best focused on the relative numerical values of k(cat). (in units of s(-1) and the substrate molarity. It is necessary only that the former be about 10(10)-10(11) times the latter to ensure that the rate of product formation be diffusion-limited and thus maximal.

scholarly journals Effect of Mg2+ and Mn2+ on isocitrate lyase, a non-essentially metal-ion-activated enzyme. A graphical approach for the discrimination of the model for activation

1991 ◽  
Vol 276 (1) ◽  
pp. 223-230 ◽  
Author(s):  
E Giachetti ◽  
P Vanni
Keyword(s):  
Kinetic Parameters ◽  
Metal Ion ◽  
Graphical Method ◽  
Isocitrate Lyase ◽  
Enzyme Reaction ◽  
Activation Mechanism ◽  
Simple Method ◽  
Suggested Approach ◽  
Graphical Approach ◽  
Substrate Complex

We describe a simple method for the analysis of activation systems in which a metal ion modifier may combine with either the enzyme or the substrate (or both) and the metal ion-substrate complex is the true substrate of the enzyme reaction. The suggested approach is essentially a ‘graphical’ method that both provides unbiased criteria for the choice of the activation mechanism and yields good rough estimates of the kinetic parameters. The procedure, tested on a variety of simulated models, produces appropriate and reliable results. Applying this treatment to isocitrate lyase, we confirmed the data previously reported for Mg2+ [Giachetti, Pinzauti, Bonaccorsi & Vanni (1988) Eur. J. Biochem. 172, 85-92], and we found that Mn2+ functions with the same mechanism as does Mg2+, but with quite different kinetic constants. In particular, its ratio of the Vmax, values of the activated and the non-activated enzyme is less than 1, and thus Mn2+ is to be considered an inhibitor rather than an activator.

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 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 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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