Enzyme activity curve

Table 4 shows the effects of thioredoxin reduction on the kinetic constants of the allosteric effectors and the substrates. The activator constant has a twofold higher apparent affinity (twofold lower 5) for the reduced enzyme form. The 3-PGA activation curve of the oxidized enzyme was slightly sigmoidal (Hill n= 1.5) while the reduced enzyme activation curve for 3-PGA was essentially hyperbolic (Hill =0.8). Thus the activity at... 

A pH-enzyme activity curve is shovm in Figure 9-1. Which of the following pairs of amino acids would be likely candidates as catalytic groups (See Primary Text, Table X-X for the pKa values of amino acid residues.)... 

FIGURE 23.12 Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate in the (a) absence and (b) presence of 25 /xM AMP. In (a) and (b), enzyme activity is plotted against substrate (fructose-1,6-bisphosphate) concentration. Concentrations of fructose-2,6-bisphosphate (in fiM) are indicated above each curve, (c) The effect of AMP (0, 10, and 25 fiM) on the inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphos-phate. Activity was measured in the presence of 10 /xM fructose-1,6-bisphosphate. 

Agarwal et al. 1978), the quantification of these specific enzymes may indicate that exposure to endosulfan has occurred. Blood tests, such as decay curves for aminopyrine in plasma, which are semiquantitative indices of liver enzyme induction, have been used successfully in the past to demonstrate enzyme induction in pesticide-exposed workers. Because numerous chemicals found at hazardous waste sites also induce these hepatic enzymes, these measurements are not specific for endosulfan exposure. However, measurements of enzyme activity, together with the detection of the parent compound or its metabolites in tissue or excreta, can be useful indicators of exposure. All of these potential biomarkers require further verification in epidemiological studies. Further studies with focus on the development of methods to separate and measure the estrogenicity of endosulfan in in vitro assays would be valuable since these assays are more sensitive and discriminative than other conventional biomarkers. Preliminary results have been presented by Sonnenschein et al. (1995). 

E I is a kinetic chimera Kj and kt are the constants characterizing the inactivation process kt is the first-order rate constant for inactivation at infinite inhibitor concentration and K, is the counterpart of the Michaelis constant. The k,/K, ratio is an index of the inhibitory potency. The parameters K, and k, are determined by analyzing the data obtained by using the incubation method or the progress curve method. In the incubation method, the pseudo-first-order constants /cobs are determined from the slopes of the semilogarithmic plots of remaining enzyme activity... 

Figure 5.9 Recovery of enzyme activity after rapid dilution as described in Figure 5.8. Curve a represents the expected behavior for a control sample that was pre-incubated and diluted in the absence of inhibitor. Curve b represents the expected behavior for a rapidly reversible inhibitor. Curve c represents the expected behavior for a slowly reversible inhibitor, and curve d represents the expected behavior for an irreversible or very slowly reversible inhibitor. See color insert.

The hallmark of slow binding inhibition is that the degree of inhibition at a fixed concentration of compound will vary over time, as equilibrium is slowly established between the free and enzyme-bound forms of the compound. Often the establishment of enzyme-inhibitor equilibrium is manifested over the time course of the enzyme activity assay, and this leads to a curvature of the reaction progress curve over a time scale where the uninhibited reaction progress curve is linear. We saw... 

From the calibration curve, the concentration of NAD+ in both the test and blank can be determined. Front the difference in the results the amount of NAD+ generated in 1 second can be calculated (mol 1 1 s l) and from this the enzyme activity in kalals per litre of sample ... 

Many automated instruments measure enzyme activity using fixed time colorimetric methods. Some, however, can be classed as reaction rate analysers, e.g. the centrifugal analysers, and these instruments determine the reaction rate from either the initial slope of the reaction curve or from repeat measurements at fixed intervals. In both methods the slope of the line is taken to represent the activity of the enzyme. 

The presence of a lag period in many coupled assays and difficulties in determining the linear portion of a curve present the main problems in the calculation of enzyme activity using reaction rate analysers. In the simplest instruments the slope of the curve in the first few seconds of the reaction is extrapolated into a straight line or, if the reaction is known to show a lag period, the rate of reaction after a defined period of time can be measured. The more sophisticated instruments use microcomputers to determine the linear portion of the curve and calculate the enzyme activity directly from the slope. The second derivative of the reaction progress curve (rate of change of the slope) can be monitored by the computer and when a value of zero is held for a period of time (10—15 seconds) this indicates a linear section of the graph. From the value for the slope, the enzyme activity can be calculated. 

An alternative approach involves the measurement of the time taken for small fixed changes in absorbance and the calculation of the rate for each single measurement. The computer stores and compares all the values, finally selecting the slope of the linear section of the curve and again calculating the enzyme activity. 

Under many circumstances, the behavior of a simple unireactant enzyme system cannot be described by the Michaelis-Menten equation, although a v versus [S] plot is still hyperbolic and can be described by a modified version of the equation. For example, as will be discussed later, when enzyme activity is measured in the presence of a competitive inhibitor, hyperbolic curve fitting with the Michaelis-Menten equation yields a perfectly acceptable hyperbola, but with a value for Km which is apparently different from that in the control curve O Figure 4-7). Of course, neither the affinity of the substrate for the active site nor the turnover number for that substrate is actually altered by the presence of a competitive... 

Irreversible inhibitors may be distinguished graphically from reversible noncompetitive inhibitors by plotting Vmax versus [E]t, where [E]t represents the total units of enzyme activity in the assay. For a noncompetitive inhibitor, the slope of the curve in the presence of inhibitor will be less than that of the control plot, and the plot will pass through the origin. If the inhibitor is instead irreversible, the slope of the curve in the presence of inhibitor will be identical to that of the control data, and the line will intersect the horizontal [E]t axis at a point equivalent to the concentration of enzyme irreversibly inactivated (Segel, 1993). 

Noncompetitive ELISA methods are based on sandwich assays in which an excess supply of immobilized primary antibody, the capture antibody, quantitatively binds the antigen of interest and an enzyme-labeled secondary antibody is then allowed to react with the bound antigen forming a sandwich. A color reaction product produced by the enzyme is then used to measure the enzyme activity that is bound to the surface of the microtiter plate. Sandwich ELISA (noncompetitive) methods yield calibration curves in which enzyme activity increases with increasing free antigen concentration. 

Figure 3.7 First and zero order components of a hyperbolic curve of enzyme activity against substrate concentration. For the first order, rate is directly proportional to concentration at zero order, rate is independent or almost independent of substrate concentration.

Hepatic elimination obeys exponential kinetics because metabolizing enzymes operate in the quasilinear region of their concentration-activity curve hence the amount of drug metabolized per unit of time diminishes with decreasing blood concentration. 

The effect of enzymes is strongly dependent on the pH value (see p.30). When the activity is plotted against pH, a bell-shaped curve is usually obtained (1). With animal enzymes, the pH optimum—i.e., the pH value at which enzyme activity is at its maximum—is often close to the pH value of the cells (i.e., pH 7). 

In characterizing the pH dependence of enzyme activity, one often observes (a) a bell-shaped curve in plots of activity versus pH (Fig. 1), or (b) S -shaped activity versus pH curves (either falling from optimal activity or rising to optimal activity) with an inflection point at some... 

Inhibition of enzyme activity by a chemical species that binds slowly and is tight-binding as well has a low dissociation constant). Such inhibitors require special kinetic analysis . The most common method of obtaining the inhibition parameters is by nonlinear regression analysis of the progress curves. 

With albumin-solubilized bilirubin, pH optima of microsomal bilirubin UDP-glucuronyltransferase were 7.4-8.0 for rat (H2, HIO, SIO) and 7.4 for guinea pig (M13) and rabbit (T8). Above pH 8 the enzyme activity decreased abruptly (HIO). In absence of carrier protein, optima were at pH 8 and 8.2 with preparations from liver of guinea pig (P3) and rat (W12), respectively. The activity-pH curve with optimum at pH 8.2 (W12) showed pronounced skewing, with a steady and rather rapid increase of enzyme activity from pH 7.4 to 8.2. One may wonder whether such measurements were influenced by the rapid increase of solubility of the acceptor substrate occurring over the same pH range (B25). 

Obviously, extrapolation procedures are impractical for routine determination of enzyme activities. When substrate saturation-curves conform to rectangular hyperbolas, reasonable concentrations of substrates should equal 10 to 20 times the respective Km values. As outlined above, application of this rule to assays of bilirubin UDP-glycosyltransferase activities is hampered by substrate inhibition and by occasional deviation from Michaelis-Menten kinetics. The best alternative in such cases may be to choose the concentrations at optimal enzyme activity. However, great care should be exercised in interpreting the results. When a bio-... 

K,y can then be obtained from a plot of v against [D] if the receptor concentration is constant. This is, of course, the same as the direct plot of enzyme activity shown in every biochemistry textbook. As with all hyperbolic relationships, there are several drawbacks to this technique many data points are needed at the beginning of the curve, at low [D] values, where accuracy is limited. Also, determination of the maximum effect is almost impossible, since we are dealing with an asymptotic curve. 

With the exception of the enzyme from the limpet, P. vulgata, a-D-mannosidase from most of the important sources shows optimal activity at pH values lying between 4 and 5. For the enzyme from jack-bean meal39 and that from rat epididymis,80 we employed a pH of 5 for routine assays. If this is not the actual optimum, it is close to it on the broad pH-activity curves, and, at this pH, the addition of Zn2+ and other cations has relatively little effect in the assay, thus simplifying the study of the various metal complexes that can be formed by the enzyme protein. 

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