Effector plots

An allosteric situation where is constant but the apparent changes in response to effectors is termed a V system. In a V system, all v versus S plots are hyperbolic rather than sigmoid (Figure 15.12). The positive heterotropic effector A activates by raising whereas 1, the negative heterotropic effec-... 

First draw both Lineweaver-Burk plots and Hanes-Woolf plots for the following a Monod-Wyman-Changeux allosteric K enzyme system, showing separate curves for the kinetic response in (1) the absence of any effectors (2) the presence of allosteric activator A and (3) the presence of allosteric inhibitor I. Then draw a similar set of curves for a Monod-Wyman-Changeux allosteric Uenzyme system. 

Figure 11. Allosteric regulation A conformational change of the active site of an enzyme induced by reversible binding of an effector molecule (A). The model of Monod, Wyman, and Changeux (B) Cooperativity in the MWC is induced by a shift of the equilibrium between the T and R state upon binding of the receptor. Note that the sequential dissociation constants Kr and KR do not change. The T and R states of the enzyme differ in their catalytic properties for substrates. Both plots are adapted from Ref. 140. See color insert.

Fig. 7. The effect of various substances on the spin-state equilibriutn of bacterial cytochrome P-450. The fraction of protein in the high-spin state is plotted (in arbitrary units) against the concentration (in fiM) of the natural effector putidaredoxin (Pd°) or the concentration in percent (v/v) of the organic solvents ethylene glycol or n-butanol. It is apparent that butanol induces a shift in spin state which is similar to that induced by the protein effector.

A substance, agent, or factor (other than a catalyst) the presence of which will increase the rate of a catalyzed reaction. A substance that activates an enzyme-catalyzed reaction by binding to the enzyme is often referred to as an enzyme activator. See Activation Allosteric Effector Linked Functions London-Steck Plot... 

Except for very simple systems, initial rate experiments of enzyme-catalyzed reactions are typically run in which the initial velocity is measured at a number of substrate concentrations while keeping all of the other components of the reaction mixture constant. The set of experiments is run again a number of times (typically, at least five) in which the concentration of one of those other components of the reaction mixture has been changed. When the initial rate data is plotted in a linear format (for example, in a double-reciprocal plot, 1/v vx. 1/[S]), a series of lines are obtained, each associated with a different concentration of the other component (for example, another substrate in a multisubstrate reaction, one of the products, an inhibitor or other effector, etc.). The slopes of each of these lines are replotted as a function of the concentration of the other component (e.g., slope vx. [other substrate] in a multisubstrate reaction slope vx. 1/[inhibitor] in an inhibition study etc.). Similar replots may be made with the vertical intercepts of the primary plots. The new slopes, vertical intercepts, and horizontal intercepts of these replots can provide estimates of the kinetic parameters for the system under study. In addition, linearity (or lack of) is a good check on whether the experimental protocols have valid steady-state conditions. Nonlinearity in replot data can often indicate cooperative events, slow binding steps, multiple binding, etc. 

ACTIVATOR ACTIVATION ALLOSTERIC EFFECTOR LINKED FUNCTIONS LONDON-STECK PLOT Active enzyme crystallography,... 

In the case of most enzymic transformations the reaction rate can be described as a hyperbolic function of the concentration of substrate the characteristic parameters of these hyperboles are the and the KM values, which can be determined easily by different linearized plots. Different factors such as temperature, pH, chemical modification of the functional groups in the side chains of the protein, reversible inhibitors, activators, allosteric effectors, influence the catalytic activity of the enzymes. 

The elasticity, e, of an enzyme is a measure of how that enzyme s catalytic activity changes when the concentration of a metabolite—substrate, product, or effector—changes. It is obtained from an experimental plot of the rate of the reaction catalyzed by the enzyme versus the concentration of the metabolite, at metabolite concentrations that prevail in the cell. By arguments analogous to those used to derive C, we can show e to be the slope of the tangent to a plot of... 

E (entgegen) configuration 43 E3 binding protein 796 Eadie-Hofctee plot 460 E-cadhedrin 574 Echinodermata 25 Ectoderm 23 Ectoenzymes 409 Ectopic proteins 573 Edelman, Gerald M. 84 Edman degradation 118 EDTA. See Ethylenediaminetetraacetic acid Effector(s) of allosteric enzymes 473-475 EF-hand motif 313, 317 EGF (epithelial growth factor), definition of 577... 

A plot of VQ against [S] for an allosteric enzyme gives a sigmoidal-shaped curve. Allosteric enzymes often have more than one active site which co-operatively bind substrate molecules, such that the binding of substrate at one active site induces a conformational change in the enzyme that alters the affinity of the other active sites for substrate. Allosteric enzymes are often multi-subunit proteins, with an active site on each subunit. In addition, allosteric enzymes may be controlled by effector molecules (activators or inhibitors) that bind to a site other than the active site and alter the rate of enzyme activity. Aspartate transcarbamoylase is an allosteric enzyme that catalyzes the committed step in pyrimidine biosynthesis. This enzyme consists of six catalytic subunits each with an active site and six regulatory subunits to which the allosteric effectors cytosine triphosphate (CTP) and ATP bind. Aspartate transcarbamoylase is feedback-inhibited by the end-product of the pathway, CTP, which acts as an allosteric inhibitor. In contrast, ATP an intermediate earlier in the pathway, acts as an allosteric activator. 

To experimentally define these kinds of interactions, it is necessary to vary both substrate and effector concentrations. For Eq. (13), initial parameters can be obtained by first performing double reciprocal plots and then replotting 1/slope and 1/intercept versus 1/[I] (21). The intercept of the 1/intercept replot is fiVmK — / ), which can be used to solve for /i. The value for a can then be obtained from the 1/slope intercept = [fjVm/Km(c/. — //). ... 

Unlike the midpoint slope (//1/2) of an ideal Nernstian plot, the slope of a non-Nernstian response cannot be interpreted as the number of electrons involved in the oxidation/reduction process. For the Hbs, the n parameter is influenced by site-site heterogeneity and allosteric effects.The n parameter is an indicator of the level of cooperativity that is operative high n values indicate a high level of cooperativity, while low n values indicate reduced cooperativity. The sensitivity of the n parameter to heterotropic effectors may be seen in Figure 2.11. The trend illustrated is consistent with the two-state (R and T) model for Hb. Maximum cooperativity is indicated by the highest values for max (defined in Figure 2.4) as illustrated for Hb o the absence of a heterotropic effector. The T-state is stabilised by heterotropic effectors (data points 1-4), which results in an increase in ease of reduction (increase in 1/2) and a decrease in cooperativity (decrease in max) due to a diminished ease of T R shift as a result of T-state stabilisation. R-state stabilisation occurs in HbCPA and horse Hb (data points 6-9), which is characterised by an increase in ease of oxidation (lower Eijf) and reduced cooperativity as illustrated by diminished max values. 

Figure 2.12 Plot of F1/2 for Hb as a function of imidazole concentration that illustrates the influence of homotropic effector equilibrium reaction 14 on the ease of reduetion and level of cooperativity (inset). Parameters obtained by spectroelectrochemistry max is defined in Figure 2.4. Conditions Pt mesh electrode [heme] = 0.1-0.23 mM [Ru(NH3)6Cl3] = 0.30-1.1 mM [NaN03] = 200mM [MOPS] = 50 mM at pH 7.1 20 °C. Figure adapted from ref. 11 and used with permission.

Liver pyruvate kinase. A plot of pyruvate kinase activity versus PEP concentration is presented. The response of activity to PEP concentration is sigmoidal. Shifts in the curve (one of an infinite number, depending on the concentration of the effectors) in the positive (F-1,6-BP and 6-PG) and negative (alanine) direction are indicated by dashed lines. The physiological range of PEP in liver is shown by the... 

Covalent modification of liver pyruvate kinase. A plot of pyruvate kinase versus PEP concentration is presented. The black solid curve indicates the response of velocity with change in PEP concentration for the nonphosphorylated enzyme in the absence of positive or negative effectors. The dashed black curves show the activity in the presence of a positive effector (6-PG) or a negative effector (alanine). The solid purple curve indicates the response of velocity with change in PEP concentration for the phosphorylated enzyme in the absence of positive or negative effectors. The dashed purple curve is the activity of the phosphorylated enzyme in the presence of a positive effector (6-PG) or a negative effector (alanine). 

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