Enzymes inhibition reactions

Fig. 4. Relationship between fluoride concentration and enzyme inhibition. Reaction mixtures contained in addition to substrate and fluoride, 0.1 M acetate, and 40-fold purified enzyme (in 0.01% gelatin), all at pH 5.5 in a 1.0-ml reaction volume. Points designated by triangles and plus symbols (+) are calculated from theory. Curve 1 /3-Glycerol-PO (13M). Curve 2 Yeast adenylic acid (0.044M). Curve 3 Phenyl-PO (0.14 M). From Reiner et al. (40).

Wall, D. B., Finch, J. W., and Cohen, S. A. (2004). Comparison of desorption/ionization on silicon (DIOS) time-of-flight and liquid chromatography/tandem mass spectrometry for assaying enzyme-inhibition reactions. Rapid Commun. Mass Spectrom. 18 1482-1486. 

In QSAR of enzyme inhibition reactions, quantum-chemically calculated electrostatic or MO-related descriptors have been widely used. The former are expected to describe the complex formation between enzyme and the substrate, whereas the latter reflect the chemical reactivity of the substrate at the site. Already in 1967, Klopman and Hudson [83] developed a polyelectronic perturbation theory, according to which the drug-receptor interactions can be under either charge or orbital control. Thus the net atomic... 

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. 

Chelation is a feature of much research on the development and mechanism of action of catalysts. For example, enzyme chemistry is aided by the study of reactions of simpler chelates that are models of enzyme reactions. Certain enzymes, coenzymes, and vitamins possess chelate stmctures that must be involved in the mechanism of their action. The activation of many enzymes by metal ions most likely involves chelation, probably bridging the enzyme and substrate through the metal atom. Enzyme inhibition may often result from the formation by the inhibitor of a chelate with a greater stabiUty constant than that of the substrate or the enzyme for a necessary metal ion. 

Enzyme Inhibition. En2yme inhibitors (qv) are reagents that bind to the enzyme and cause a decrease in the reaction rate. Irreversible inhibitors bind to the enzyme by an irreversible reaction, and consequendy cannot dissociate from the enzyme or be removed by dilution or dialysis. Examples of irreversible inhibitors are nerve gases such as diisopropylphosphoduoridate [55-91-4] (DEP). 

Inhibition The decrease of the rate of an enzyme-catalyzed reaction by a chemical compound including substrate analogues. Such inhibition may be competitive with the substrate (binding at die active site of die enzyme) or non-competitive (binding at an allosteric site). 

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. 

In general, enzymes are proteins and cany charges the perfect assumption for enzyme reactions would be multiple active sites for binding substrates with a strong affinity to hold on to substrate. In an enzyme mechanism, the second substrate molecule can bind to the enzyme as well, which is based on the free sites available in the dimensional structure of the enzyme. Sometimes large amounts of substrate cause the enzyme-catalysed reaction to diminish such a phenomenon is known as inhibition. It is good to concentrate on reaction mechanisms and define how the enzyme reaction may proceed in the presence of two different substrates. The reaction mechanisms with rate constants are defined as ... 

Toxin (Enzyme Inhibition) Biosensors Enzyme affectors (inhibitors and activators) that influence the rate of biocatalytic reactions can also be measured. Sensing probes for organophosphate and carbamate pesticides, for the respiratory... 

One form of biological poisoning mirrors the effect of lead on a catalytic converter. The activity of an enzyme is destroyed if an alien substrate attaches too strongly to the enzyme s active site, because then the site is blocked and made unavailable to the true substrate (Fig. 13.42). As a result, the chain of biochemical reactions in the cell stops, and the cell dies. The action of nerve gases is believed to stem from their ability to block the enzyme-controlled reactions that allow impulses to travel through nerves. Arsenic, that favorite of fictional poisoners, acts in a similar way. After ingestion as As(V) in the form of arsenate ions (As043 ), it is reduced to As(III), which binds to enzymes and inhibits their action. 

Information relevant to the mechanism of an enzyme-catalyzed reaction can, in general, only be obtained from irreversible inhibitors which react specifically at the active site and thereby inactivate the enzyme. As active-site-directed inhibition is treated in detail in Ref. 142 general aspects will be discussed here only briefly. In order to be suitable as an active-site-directed inhibitor, a compound must fulfil the following requirements. 

Chohnesterase-inhibiting pesticides (e g., organophosphate and carbamate pesticides) are detected by dipping the developed chromatogram in a solution of the enzyme chohnesterase followed by incubation for a short period. Then the plate is dipped in a substrate solution, e.g., 1-naphthyl acetate/fast blue salt B. In the presence of the active enzyme, 1-naphthyl acetate is hydrolyzed to 1-naphthol and acetic acid, and the 1-naphthol is coupled with fast blue salt B to form a violet-blue azo dye. The enzyme is inhibited by the pesticide zones, so the enzyme-substrate reaction does not occur pesticides are, therefore, detected as colorless zones on a violet-blue background [36]. 

Determination of the IC50 is a preliminary evaluation of the relative affinity of different compounds for a target enzyme. To evaluate affinity properly, however, one must first define the mechanism of inhibition of the target enzyme by each compound. The next step in the lead evaluation flowchart (Figure 5.1) is to determine if the inhibition caused by a compound is rapidly reversible, slowly reversible, or irreversible. This information will help the investigator understand whether or not the inhibition reaction can be treated as a reversible equilibrium, and thus decide on the best measure of true affinity for a particular compound. 

Inhibition Effects in Enzyme Catalyzed Reactions. Enzyme catalyzed reactions are often retarded or inhibited by the presence of species that do not participate in the reaction in question as well as by the products of the reaction. In some cases the reactants themselves can act as inhibitors. Inhibition usually results from the formation of various enzyme-inhibitor complexes, a situation that decreases the amount of enzyme available for the normal reaction sequence. The study of inhibition is important in the investigation of enzyme action. By determining what compounds behave as inhibitors and what type of kinetic patterns are followed, it may be possible to draw important conclusions about the mechanism of an enzyme s action or the nature of its active site. 

The design and implementation of a portable fiber-optic cholinesterase biosensor for the detection and determination of pesticides carbaryl and dichlorvos was presented by Andreou81. The sensing bioactive material was a three-layer sandwich. The enzyme cholinesterase was immobilized on the outer layer, consisting of hydrophilic modified polyvinylidenefluoride membrane. The membrane was in contact with an intermediate sol-gel layer that incorporated bromocresol purple, deposited on an inner disk. The sensor operated in a static mode at room temperature and the rate of the inhibited reaction served as an analytical signal. This method was successfully applied to the direct analysis of natural water samples (detection and determination of these pesticides), without sample pretreatment, and since the biosensor setup is fully portable (in a small case), it is suitable for in-field use. 

Mainly, two principles are used in electrochemical pesticide biosensor design, either enzyme inhibition or hydrolysis of pesticide. Among these two approaches inhibition-based biosensors have been widely employed in analysis due to the simplicity and wide availability of the enzymes. The direct enzymatic hydrolysis of pesticide is also extremely attractive for biosensing, because the catalytic reaction is superior and faster than the inhibition [27],... 

Cyclodextrins as catalysts and enzyme models It has long been known that cyclodextrins may act as elementary models for the catalytic behaviour of enzymes (Breslow, 1971). These hosts, with the assistance of their hydroxyl functions, may exhibit guest specificity, competitive inhibition, and Michaelis-Menten-type kinetics. All these are characteristics of enzyme-catalyzed reactions. 

This is not a completely true statement. As you may see later on, the velocity of an enzyme-catalyzed reaction depends on the concentration of substrate only when the substrate concentration is near the Km. If we start out with a concentration of substrate that is 1000 times the Km, most of the substrate will have to be used up before the velocity falls because of a decrease in substrate concentration. If the product of the reaction does not inhibit and the enzyme is stable, the velocity will remain constant for much more than 1 to 5 percent of the reaction. It s only when we re near the Km that substrate depletion during the assay is a problem. 

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