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 N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors

2021 ◽  
Vol 49 (5) ◽  
pp. 2684-2699
Author(s):  
Ka-Weng Ieong ◽  
Gabriele Indrisiunaite ◽  
Arjun Prabhakar ◽  
Joseph D Puglisi ◽  
Måns Ehrenberg
Keyword(s):  
Single Molecule ◽  
Stop Codon ◽  
Product Formation ◽  
Release Factors ◽  
Substrate Complex ◽  
Peptidyl Transfer ◽  
Enzyme Substrate ◽  
Peptide Release ◽  
Accuracy Ratio ◽  
Codon Reading

Abstract We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km.

scholarly journals Proofreading through spatial gradients

2020 ◽  
Author(s):  
Vahe Galstyan ◽  
Kabir Husain ◽  
Fangzhou Xiao ◽  
Arvind Murugan ◽  
Rob Phillips
Keyword(s):  
Error Correction ◽  
Reaction Rates ◽  
Structural Features ◽  
Product Formation ◽  
Concentration Gradients ◽  
Nucleotide Hydrolysis ◽  
Substrate Complex ◽  
Spatial Gradients ◽  
Enzyme Substrate ◽  
Kinetic Proofreading

Key enzymatic processes in biology use the nonequilibrium error correction mechanism called kinetic proofreading to enhance their specificity. Kinetic proofreading typically requires several dedicated structural features in the enzyme, such as a nucleotide hydrolysis site and multiple enzyme–substrate conformations that delay product formation. Such requirements limit the applicability and the adaptability of traditional proofreading schemes. Here, we explore an alternative conceptual mechanism of error correction that achieves delays between substrate binding and subsequent product formation by having these events occur at distinct physical locations. The time taken by the enzyme–substrate complex to diffuse from one location to another is leveraged to discard wrong substrates. This mechanism does not require dedicated structural elements on the enzyme, making it easier to overlook in experiments but also making proofreading tunable on the fly. We discuss how tuning the length scales of enzyme or substrate concentration gradients changes the fidelity, speed and energy dissipation, and quantify the performance limitations imposed by realistic diffusion and reaction rates in the cell. Our work broadens the applicability of kinetic proofreading and sets the stage for the study of spatial gradients as a possible route to specificity.

scholarly journals The Cholinesterase of Erythrocytes in Anemias

Blood ◽  
1951 ◽  
Vol 6 (2) ◽  
pp. 151-159 ◽  
Author(s):  
JEAN SABINE
Keyword(s):  
Pernicious Anemia ◽  
Dissociation Constants ◽  
Relative Concentration ◽  
Enzymic Activity ◽  
Red Cells ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Bone Marrow Function ◽  
Experimental Values ◽  
Limiting Velocity

Abstract 1. Activity-pS curves were determined experimentally for the cholinesterase of the erythrocytes in various human anemias. 2. The data were analyzed to give experimental values for the apparent dissociation constants, K8 of the active enzyme-substrate complex (ES) and K2 of the inactive complex (ES2), and the theoretic limiting velocity V. 3. The analysis showed that the enzyme in the cells of anemia is the same as that in normal cells. The abnormally high enzymic activity (initial velocity at a single substrate concentration) known to occur in various anemias and the change from low activity in pernicious anemia in relapse to high activity during treatment are attributable to changes in concentration of the enzyme. 4. The enzyme in the red cells of pernicious anemia in relapse was not activated by folic acid or liver extract. 5. It was shown experimentally that the reticulocytes and young cells of normal blood and of pernicious anemia during treatment contain much higher concentrations of enzyme than old cells. The increased concentration persists for some time after the reticulum has disappeared. 6. The relative concentration of enzyme in the red cells is a more sensitive indicator of hyperactive hematopoiesis than the reticulocyte count. 7. Evidence is presented that failure of elevation of enzymic concentration in severe anemia is associated specifically with suppression or malfunction of the hematopoietic system. 8. It is believed that the relative concentration of enzyme can be used as a test of bone marrow function in severe anemias. It is hoped that further investigation may result in a useful test of wider application.

Effect of Prostaglandins (E2 and A2) on the Enzymatic Reaction of Human Renin in Isolated Homologous System and with Added Normal and Hypertensive Plasma

1974 ◽  
Vol 48 (s2) ◽  
pp. 307s-309s
Author(s):  
P. Eggena ◽  
J. Barrett ◽  
M. Sambhi
Keyword(s):  
Prostaglandin E2 ◽  
Substrate Concentration ◽  
Enzymatic Reaction ◽  
Product Formation ◽  
Inhibitory Effect ◽  
Renin Substrate ◽  
Enzyme Substrate ◽  
Normal Plasma ◽  
Human Renin ◽  
Prostaglandin A2

1. Prostaglandin E2 significantly inhibits the renin reaction in whole plasma as well as in the isolated system of semi-purified human renin and human renin substrate. The inhibitory effect of prostaglandin A2 was less marked in whole plasma and absent in the isolated system. 2. The inhibitory effect of prostaglandin E2 was more marked in normal than hypertensive plasma and was maximal at the lowest concentration used. In hypertensive plasma the maximal inhibitory effect was achieved at tenfold higher concentrations. 3. In normal plasma prostaglandin E2 does not affect the rate of product formation (k5 = k6), but inhibits the overall renin reaction by decreasing the total amount of available enzyme-substrate and enzyme-substrate modifier complex (K2K3). 4. In hypertensive plasma prostaglandin E2 acts as a potential accelerator of the rate of product formation (k6k5). In the range of substrate concentration employed, the apparent inhibitory effect is explained by an even greater lack of available complex (K2K3). This behaviour in hypertensive plasma is consistent with the presence of an additional modifier (? activator).

scholarly journals Catalytic properties of alkaline phosphatase from pig kidney

1974 ◽  
Vol 141 (1) ◽  
pp. 283-291 ◽  
Author(s):  
Kunio Hiwada ◽  
Ernst D. Wachsmuth
Keyword(s):  
Alkaline Phosphatase ◽  
Enzymic Activity ◽  
Ph Optimum ◽  
Active Centre ◽  
Substrate Complex ◽  
Pig Kidney ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

The enzymic properties of alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes were studied. 1. It hydrolyses ortho- and pyro-phosphate esters, the rate limiting step (Vmax.) being independent of the substrate. It transphosphorylates to Tris at concentrations above 0.1m-Tris. 2. The pH optimum for hydrolysis was between 9.8 and 10. The pK of the enzyme–substrate complex is 8.7 for p-nitrophenyl phosphate and β-glycerophosphate. Excess of substrate inhibits the enzymic activity with decreasing pH. The pK of the substrate-inhibited enzyme–substrate complex, 8.7, is very similar to that for the enzyme–substrate complex. The pK values of the free enzyme appear to be 8.7 and 7.9. 3. Inactivation studies suggest that there is an essential tyrosine residue at the active centre of the enzyme. 4. The energy of activation (E) and the heat of activation (ΔH) at pH9.5 showed a transition at 24.8°C that was unaffected by Mg2+. 5. Kinetic and atomic-absorption analysis indicated the essential role of two Zn2+ ions/tetrameric enzyme for an ordered association of the monomers. Zn2+ in excess and other bivalent ions compete for a second site with Mg2+. Mg2+ enhances only the rate-limiting step of substrate hydrolysis. 6. Amino acid inhibition studies classified the pig kidney enzyme as an intermediate type of previously described alkaline phosphatases. It has more similarity with the enzyme from liver and bone than with that from placenta.

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.

scholarly journals Derivable Equations and Issues Often Ignored in the Original Michaelis-Menten Mathematical Formalism

2019 ◽  
pp. 1-13
Author(s):  
Ikechukwu I. Udema
Keyword(s):  
Rate Constant ◽  
Product Formation ◽  
Theoretical Research ◽  
Quasi Steady State ◽  
Substrate Complex ◽  
The Core ◽  
Enzyme Substrate ◽  
Reverse Rate Constant ◽  
Core Issue

Background: There has been recent shift from the core issue of Michaelian kinetics to issues regarding various kinds of quasi-steady-state assumptions. Derivable equations with which to determine reverse rate constant for the dissociation of enzyme-substrate complex (ES) is given less attention. Objectives: The objectives of this research are: 1) to derive other equations from differential equations whose evaluation leads to MM equation and 2) quantify based on derived equations the kinetic parameters given less attention and duration of catalytic events. Methods: A major theoretical research and experimentation using Bernfeld method. Results and Discussion: The durations for ES dissociation (ESD) into free substrate, S and enzyme, E were much shorter than the duration of ESD into E and product, P in 3 minutes duration of assay with low [S]; it was the shortest and longest in 3 and 5 minutes durations respectively with high [S]. The durations of ESD into E and P was shortest in 3 minutes duration of assay with high [S]. The values of reverse rate constant, k-1 for ESD into S and E in 3 minutes duration of assay with high [S] was » the rate constant, k2 for product formation and they are much higher than in other duration of assay. Conclusion: The equations for the determination of the durations of various events, in a given catalytic cycle were derived. The various time regimes for each event and the rate constant for the dissociation of the ES can be graphically and calculationally determined as the case may be. Substrate concentration regime and duration of assay affects rate constants.

scholarly journals Proofreading through spatial gradients

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Vahe Galstyan ◽  
Kabir Husain ◽  
Fangzhou Xiao ◽  
Arvind Murugan ◽  
Rob Phillips
Keyword(s):  
Error Correction ◽  
Reaction Rates ◽  
Structural Features ◽  
Product Formation ◽  
Nucleotide Hydrolysis ◽  
Structural Requirements ◽  
Substrate Complex ◽  
Spatial Gradients ◽  
Enzyme Substrate ◽  
Kinetic Proofreading

Key enzymatic processes use the nonequilibrium error correction mechanism called kinetic proofreading to enhance their specificity. The applicability of traditional proofreading schemes, however, is limited since they typically require dedicated structural features in the enzyme, such as a nucleotide hydrolysis site or multiple intermediate conformations. Here, we explore an alternative conceptual mechanism that achieves error correction by having substrate binding and subsequent product formation occur at distinct physical locations. The time taken by the enzyme-substrate complex to diffuse from one location to another is leveraged to discard wrong substrates. This mechanism does not have the typical structural requirements, making it easier to overlook in experiments. We discuss how the length scales of molecular gradients dictate proofreading performance, and quantify the limitations imposed by realistic diffusion and reaction rates. Our work broadens the applicability of kinetic proofreading and sets the stage for studying spatial gradients as a possible route to specificity.

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.

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