Enzyme kinetics inhibitor

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. 

Enzyme inhibitors are classified in several ways. The inhibitor may interact either reversibly or irreversibly with the enzyme. Reversible inhibitors interact with the enzyme through noncovalent association/dissociation reactions. In contrast, irreversible inhibitors usually cause stable, covalent alterations in the enzyme. That is, the consequence of irreversible inhibition is a decrease in the concentration of active enzyme. The kinetics observed are consistent with this interpretation, as we shall see later. 

Inhibitors of the catalytic activities of enzymes provide both pharmacologic agents and research tools for study of the mechanism of enzyme action. Inhibitors can be classified based upon their site of action on the enzyme, on whether or not they chemically modify the enzyme, or on the kinetic parameters they influence. KineticaUy, we distinguish two classes of inhibitors based upon whether raising the substrate concentration does or does not overcome the inhibition. 

The Influence of Environmental Factors on Enzyme Kinetics. Because enzymes are proteins, they are unusually sensitive to changes in their environment. This is true not only with regard to variations in inhibitor concentrations, but also with respect to variations in pH and temperature. Most enzymes are efficient catalysts only within relatively narrow ranges of pH and temperature. 

Volume 63. Enzyme Kinetics and Mechanism (Part A Initial Rate and Inhibitor Methods)... 

Measurement of blue and green fluorescence of NADH and FAD in living tissues Quantitative fluorescent cytochemistry Using permeable fluorogenic substrates of enzymes, specific inhibitors, and kinetic analysis... 

Table 9.2 Summary of enzyme kinetic parameters and inhibitor potencies for 11 human CYP activities in pooled human liver microsomes [117]. 

Enzyme kinetics and determination of Kt in a buffer system (the inhibitor approach)... 

Introductions to enzyme kinetics and bioenergetics are given with explanations of key terms such as Km and Vmax coenzymes, cofactors and inhibitors typical metabolic reactions free energy exergonic and endergonic reactions, catabolism and anabolism. 

Enzyme kinetics Michaelis constant, symbol iCm maximum velocity of an enzyme catalysed reaction, Vm DC inhibitor constant, symbol X Michaelis-Menten equation and graph in the absence and the presence of inhibitors. Lineweaver-Burke and Eadie-Hofstee plots. 

Source. Casidy R, Frey R. Drug Strategies to Target HIV Enzyme Kinetics and Enzyme Inhibitors, Washington University. http //wunmr.wustl.edu/EduDev/Lab I itorials/F[IV/ DrugStrategies.html [accessed May 21,2003]. 

Abstract Neuroscientists may wish to quantify an enzyme activity for one of many reasons. In order to do so, the researcher must be able to set up an assay appropriately, and this requires some understanding of the kinetic behavior of the enzyme toward the substrate used. Furthermore, such an understanding is vital if the inhibitory effects of a drug are to be assessed appropriately. This chapter outlines key principles that must be adhered to, and describes basic approaches by which rather complex kinetic data might be obtained, in order that enzyme kinetics and inhibitor kinetics might be studied successfully by the nonexpert. 

Many substances can affect metabolic processes by influencing the activity of enzymes. Enzyme inhibitors are particularly important here. A large proportion of medicines act as enzyme inhibitors. Enzyme-kinetic experiments are therefore an important aspect of drug development and testing procedures. Natural metabolites are also involved in regulatory processes as inhibitors (see p.ll4). 

While requiring the availability of competitive inhibitors for each of the substrates, Fromm s use of competitive inhibitors to distinguish multisubstrate enzyme kinetic pathways represents the most powerful initial rate method. See Alternative Substrate Inhibition... 

Figure 8.4 The Lineweaver-Burk plot (A) and the Hanes plot (B) of typical enzyme kinetics in presence of a competitve (a) noncompetive (b), mixed type (c) and uncompetitive (d) inhibitor.

Since the four yeast PGK inhibitors are commercially available it was logical to test them for T. brucei PGK inhibition. The first three compounds were active in the millimolar range. However, SPADNS exhibited a K of 10.0 pMin these in preliminary tests [88]. Moreover, when assayed against a commercially available rabbit muscle PGK, SPADNS had no influence on the enzyme kinetics up to a concentration of 250 pM[88], In conclusion, SPADNS appears to be an excellent lead because of its potency and selectivity. Crystallographic experiments to determine its binding mode to I brucei VGK are underway. 

Enzyme kinetic studies of inhibitor are very important for considering as a therapeutic agent. It is interesting to note that isoprenoid-substituted flavonoids having non-steroidal structures are potent un-competitive inhibitors of 5a-reductase. So, it would be expected that the isoprenoid-substituted flavonoid derivative would be an interesting lead compounds for testosterone 5a-reductase inhibitor. 

Inhibition may be incorporated into the mechanism of micellar catalysis in the same way it is handled in enzyme kinetics. Representing the inhibitor by /, we can revise Reaction (G) as follows ... 

Concluding remarks. Thus most species differences in metabolism are quantitative rather than qualitative only occasionally does a particular single species show an inability to carry out a particular reaction, or to be its sole exponent. The more common quantitative differences depend on species differences in the enzyme concentration or its kinetic parameters, the availability of cofactors, the presence of reversing enzymes or inhibitors, and the concentration of substrate in the tissue. 

The homogeneous hydrogenation systems discussed in this paper may be treated as analogues of enzyme systems with the rhodium catalyst as the enzyme (E), hydrogen (Si) and cyclohexene (S2) as the substrates, and excess ligand or other donor site as the inhibitor (I). The well-established mathematical operations of enzyme kinetics (12) can then be used to derive rate equations for various possible mechanisms. 

A Lineweaver-Burk plot of enzyme kinetics in the presence and absence of a noncompetitive inhibitor is shown in Figure E5.5. Umax in the presence of a noncompetitive inhibitor is decreased, but KM is unaffected. The effect of a competitive inhibitor on the direct linear plot is shown in Figure E5.6. 

Inhibition kinetics are included in the second category of assay applications. An earlier discussion outlined the kinetic differentiation between competitive and noncompetitive inhibition. The same experimental conditions that pertain to evaluation of Ku and Vmax hold for A) estimation. A constant level of inhibitor is added to each assay, but the substrate concentration is varied as for Ku determination. In summary, a study of enzyme kinetics is approached by measuring initial reaction velocities under conditions where only one factor (substrate, enzyme, cofactor) is varied and all others are held constant. 

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