Enzymes, inhibition, substrate effect

The three reversible mechanisms for enzyme inhibition are distinguished by observing how changing the inhibitor s concentration affects the relationship between the rate of reaction and the concentration of substrate. As shown in figure 13.13, when kinetic data are displayed as a Lineweaver-Burk plot, it is possible to determine which mechanism is in effect. 

Enzyme reaction kinetics were modelled on the basis of rapid equilibrium assumption. Rapid equilibrium condition (also known as quasi-equilibrium) assumes that only the early components of the reaction are at equilibrium.8-10 In rapid equilibrium conditions, the enzyme (E), substrate (S) and enzyme-substrate (ES), the central complex equilibrate rapidly compared with the dissociation rate of ES into E and product (P ). The combined inhibition effects by 2-ethoxyethanol as a non-competitive inhibitor and (S)-ibuprofen ester as an uncompetitive inhibition resulted in an overall mechanism, shown in Figure 5.20. 

To refer to the kinetics of allosteric inhibition as competitive or noncompetitive with substrate carries misleading mechanistic implications. We refer instead to two classes of regulated enzymes K-series and V-se-ries enzymes. For K-series allosteric enzymes, the substrate saturation kinetics are competitive in the sense that is raised without an effect on V. For V-series allosteric enzymes, the allosteric inhibitor lowers... 

A reciprocal plot of the effect of varying concentrations of a noncompetitive inhibitor on enzyme-catalyzed substrate turnover readily reveals the nature and characteristics of this type of inhibition (Fig. 3.6). Notice that in this case, the properties that characterize noncompetitive inhibition are virtually opposite of those that characterize competitive inhibition. With a noncompetitive inhibitor Emax does change but KM is constant. 

In partial (hyperbohc) mixed inhibition (O Figure 4-12d), binding of inhibitor to a site distinct from the active site results in altered affinity of enzyme for substrate (by a factor, ot) as well as a change (by a factor, /i) in the rate at which product can be released from ESI. The effects of a partial mixed inhibitor on a Lineweaver-Burk plot depend upon the actual values, and on the relative values, of ot and fl. Once again, inhibitor plots can intersect the control plot above or below, but not on, the oeaxis, and to the left or to the right of, but not on, the y-axis. Because Vmax cannot be driven to zero, a maximum Lineweaver-Burk slope is reached at infinitely high inhibitor concentrations beyond which no further increase occurs. 

Enzyme inhibition by an extremely tight-binding inhibi-tor When the substrate(s), regardless of the detailed mode of inhibition, has (have) a negligible effect on the formation of enzyme-inhibitor (E-I) complex, the net result is depletion i.e., the removal of enzyme by the inhibitor from the reaction). The observed kinetic pattern is identical to the simple noncompetitive inhibition case the substrate and the inhibitor do not affect each other s binding, because only V sk is changed due to reduced enzyme concentration, while remains unaltered. 

Product inhibition and substrate inhibition are effects also known in enzyme catalysis that can reduce catalytic efficiency. Generally, catalytic systems (natural or artificial) based on covalent interactions are more sensitive towards inhibitions than non-covalent systems utilizing weak interactions Garcia-Junceda, E. (2008) Multi-Step Enzyme Catalysis, Wiley-VCH Verlag GmbH, Weinheim, Germany. 

The answer is D. Organophosphates react with the active site serine residue of hydrolases such as acetylcholinesterase and form a stable phosphoester modification of that serine that inactivates the enzyme toward substrate. Inhibition of acetylcholinesterase causes overstimulation of the end organs regulated by those nerves. The symptoms manifested by this patient reflect such neurologic effects resulting from the inhalation or skin absorption of the pesticide diazinon. 

Mirtazapine is a substrate for several CYP450 enzymes including 2D6, 3A4, and 1A2. Consequently, drugs that inhibit these isozymes may raise mirtazapine levels. However, mirtazapine is not an inhibitor of these enzymes. The sedating effects of mirtazapine may be additive with those of CNS depressants such as alcohol and benzodiazepines. 

Enzyme inhibition. The enzymes of biotransformation may be inhibited by a single exposure to chemicals. This occurs by several mechanisms formation of a complex, competition between substrates, destruction of the enzyme, reduced synthesis of the enzyme, allosteric effects, and lack of cofactors. The consequences will depend on the role of metabolism in toxicity in the same way as induction (see above). 

Since a competitive inhibitor has a strong structural resemblance to the substrate, both the inhibitor and substrate compete for the active site of an enzyme. The formation of an enzyme-inhibitor complex reduces the amount of enzyme available for interaction with the substrate and, as a result, the rate of reaction decreases. A competitive inhibitor normally combines reversibly with enzyme. Therefore, the effect of the inhibitor can be minimized by increasing the substrate concentration, unless the substrate concentration is greater than the concentration at which the substrate itself inhibits the reaction. The mechanism of competitive inhibition can be expressed as follows ... 

Noncompetitive inhibitors can bind to both the enzyme and enzyme-substrate complex to form either an enzyme-inhibitor complex or an enzyme-inhibitor-substrate complex. The net result is a decrease in Vmax but no change in Km. Metyrapone (Figure 9.6), a well-known inhibitor of monooxygenase reactions, can also, under some circumstances, stimulate metabolism in vitro. In either case the effect is noncompetitive, in that the Km does not change, whereas Vm. does, decreasing in the case of inhibition and increasing in the case of stimulation. 

The biochemical aspects of teratology are not particularly well understood. Several kinds of biochemical mechanisms are probably involved. One such mechanism is interference with DNA synthesis, which alters the function of nucleic acids in cell replications, resulting in effects that are expressed as birth defects. Exposure to teratogenic xenobiotic substances may result in either an absence or excess of chromosomes. Enzyme inhibition (see Section 7.6) by xenobiotics can result in birth defects. Xenobiotics that deprive the fetus of essential substrates (for example, vitamins), that interfere with energy supply, or that alter the permeability of the placental membrane may all cause birth defects. 

Enzyme immobilization has been reported to improve the thermal stability of enzymes (1,2) and may also affect binding of substrates and inhibitors to the enzyme, thereby affecting the Michaelis constant and enzyme inhibition. Several previous studies have considered the advantages of immobilized enzymes with soluble substrates, and a few studies have also investigated the properties of immobilized enzymes with insoluble substrates. The main objective of the present work was to establish the effect of immobilization on the thermal stability of these enzymes, so that they may be used at elevated temperatures without significant activity loss. The immobilization conditions were varied, and their effect on the performance of the immobilized enzymes was analyzed with reference to their physiochemical and structural properties. 

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