Enzymatic velocities

FIGURE 2.2 Fluorescence intensity readout principle. In the intact peptidic substrate (amino acids symbolized by X, Y, and Z) labeled with a fluorophore at the C terminus, the intensity of fluorescence emission (light gray arrow) after excitation (dark gray arrow) is low. Through the cleavage of the substrate between the C terminal amino acid (Z) and the fluorophore by a protease, the intensity of fluorescence emission is strongly enhanced. An increase of fluorescence intensity over time dependent on the enzymatic velocity is observed. 

The Michaelis constant, KM, can be obtained from measurements conducted at a constant enzyme concentration and different substrate concentrations. Under conditions in which the substrate concentration [S] is significantly higher than the enzyme concentration and the substrate consumption is below 20%, the initial enzymatic velocity v0 can be approximated by the Henri-Michaelis-Menten equation ... 

Thus, when [S] << Km, the enzymatic velocity depends on the values of fccat/KM, [S], and [E].. Under these conditions, fecat/ M is the rate constant for the interaction of S and E. The rate constant fecat/ M i measure of catalytic efficiency because it takes into account both the rate of catalysis with a particular substrate (fecat) strength of the enzyme—substrate inter-... 

Kluge and Osmond, 1972 Ting and Osmond, 1973 a, b). Because of endogenous inhibitors, diurnal and seasonal fluctuations of enzymic activity, and environment-dependent shifts in metabolic pathways, maximum enzymatic velocities in succulent plants are difficult to obtain and awkward to evaluate. Nevertheless, maximum velocity estimates are high and in the C4 plant range of 30pmol min" and far above the estimate of 1.5 pmol min" mg chlorophyll" of C3 plants. 

FIGURE 12.12 (A) Initial (non-normalized) enzymatic velocities in citrate buffer solu-... 

Abbreviations K Michaelis constant maximal enzymatic velocity Ki, product inhibition constant. 

It is essential to maintain high, maximal velocities of enzymatic activity for the attainment of optimal therapeutic efficacy. As a general rule, only enzymes whose MichaeHs-Menten constants He between 1—100 ]lM are effective as dmgs (16) because most substrates for therapeutically useful enzymes are present ia body fluids and cells at suhmillimolar concentrations. 

The three most common types of inhibitors in enzymatic reactions are competitive, non-competitive, and uncompetitive. Competitive inliibition occurs when tlie substrate and inhibitor have similar molecules that compete for the identical site on the enzyme. Non-competitive inhibition results in enzymes containing at least two different types of sites. The inhibitor attaches to only one type of site and the substrate only to the other. Uncompetitive inhibition occurs when the inhibitor deactivates the enzyme substrate complex. The effect of an inhibitor is determined by measuring the enzyme velocity at various... 

Saturation kinetics are also called zero-order kinetics or Michaelis-Menten kinetics. The Michaelis-Menten equation is mainly used to characterize the interactions of enzymes and substrates, but it is also widely applied to characterize the elimination of chemical compounds from the body. The substrate concentration that produces half-maximal velocity of an enzymatic reaction, termed value or Michaelis constant, can be determined experimentally by graphing r/, as a function of substrate concentration, [S]. 

Kinetics is the branch of science concerned with the rates of chemical reactions. The study of enzyme kinetics addresses the biological roles of enzymatic catalysts and how they accomplish their remarkable feats. In enzyme kinetics, we seek to determine the maximum reaction velocity that the enzyme can attain and its binding affinities for substrates and inhibitors. Coupled with studies on the structure and chemistry of the enzyme, analysis of the enzymatic rate under different reaction conditions yields insights regarding the enzyme s mechanism of catalytic action. Such information is essential to an overall understanding of metabolism. 

If the velocity of an enzymatic reaction is decreased or inhibited, the kinetics of the reaction obviously have been perturbed. Systematic perturbations are a basic tool of experimental scientists much can be learned about the normal workings of any system by inducing changes in it and then observing the effects of the change. The study of enzyme inhibition has contributed significantly to our understanding of enzymes. 

Similarly to quantitative determination of high surfactant concentrations, many alternative methods have been proposed for the quantitative determination of low surfactant concentrations. Tsuji et al. [270] developed a potentio-metric method for the microdetermination of anionic surfactants that was applied to the analysis of 5-100 ppm of sodium dodecyl sulfate and 1-10 ppm of sodium dodecyl ether (2.9 EO) sulfate. This method is based on the inhibitory effect of anionic surfactants on the enzyme system cholinesterase-butyryl-thiocholine iodide. A constant current is applied across two platinum plate electrodes immersed in a solution containing butyrylthiocholine and surfactant. Since cholinesterase produces enzymatic hydrolysis of the substrate, the decrease in the initial velocity of the hydrolysis caused by the surfactant corresponds to its concentration. Amounts up to 60 pg of alcohol sulfate can be spectrometrically determined with acridine orange by extraction of the ion pair with a mixture 3 1 (v/v) of benzene/methyl isobutyl ketone [271]. 

The mechanical behavior of the contractile apparatus of smooth muscle is also very similar to that of striated muscle. So that to the extent that the force-velocity curves reflect the interaction of mechanical force and the rate of enzymatic catalysis, the steps of the chemomechanical transduction cycles in the two muscles are apparently modulated in similar ways. Also relationships between the active isometric force and muscle length are very similar (except as noted above for shorter lengths). 

Little is known about the substrate specificities of most of the enzymes as compared with common reagents used in organic synthesis. The velocity of the enzymatic... 

We consider the initial velocities V, observed with different substrate concentrations X in a rate-limited enzymatic reaction [15] ... 

The initial velocity of reaction is defined by the slope of a linear plot of product (or substrate) concentration as a function of time (Chapter 2), and we have just discussed the importance of measuring enzymatic activity during this initial velocity phase of the reaction. The best measure of initial velocity is thus obtained by continuous measurement of product formation or substrate disappearance with time over a convenient portion of the intial velocity phase. However, continuous monitoring of assay signal is not always practical. Copeland (2000) has described three types of assay readouts for measuring reaction velocity continuous assays, discontinuous... 

Figure 4.6 Reaction velocity as a function of enzyme concentration for a non-ideal enzymatic activity assay. Note the deviations from the expected linear relationship at low and at high enzyme concentration.

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

Figure 6.2 Effect of preincubation time with inhibitor on the steady state velocity of an enzymatic reaction for a very slow binding inhibitor. (A) Preincubation time dependence of velocity in the presence of a slow binding inhibitor that conforms to the single-step binding mechanism of scheme B of Figure 6.3. (B) Preincubation time dependence of velocity in the presence of a slow binding inhibitor that conforms to the two-step binding mechanism of scheme C of Figure 6.3. Note that in panel B both the initial velocity (y-intercept values) and steady state velocity are affected by the presence of inhibitor in a concentration-dependent fashion.

Figure 6.19 Fractional velocity for the enzymatic reaction of COX2 as a function of preincubation time with varying concentrations of the slow binding inhibitor DuP697. The lines drawn through the data represent the best fits to Equation (6.4).

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