Irreversible enzyme inhibition method

Irreversibility of inhibition can be established in a number of ways. Basically, excess inhibitor must be removed from the enzyme to isolate the possible reactivation process and enzyme activity monitored with time to test for any reactivation. Methods include exhaustive dialysis of inhibited enzyme with uninhibited enzyme as a control, removing aU excess inhibitor and allowing time for reactivation, followed by assay for activity. An incubation of enzyme and inhibitor followed by dilution into assay solution will measure spontaneous recovery. The stability of the enzyme adduct to exogenous nucleophiles can be determined by diluting the incubation mixture into a solution containing an exogenous nucleophile, such as S-mercaptoethanol or hydroxylamine. Gel filtration or fast filtration columns also effectively remove inhibitor, and activity assays of the protein fraction can monitor any reactivation of the enzyme-inhibitor complex. 

A method has been described by which the effects of reversible competitive monoamine oxidase inhibitors might be estimated successfully ex vivo (Green, 1984). This method relies upon the ability of a reversible competitive inhibitor (perhaps administered chronically) to protect against the effects of an irreversible inhibitor (administered as a single dose) that binds to the enzyme active site. As inhibition by an irreversible inhibitor can be measured quite easily ex vivo, the degree of irreversible inhibition in an animal coadministered a reversible competitive inhibitor would be less than that in a control animal that received only the irreversible inhibitor. The difference would provide an estimate of the degree to which enzyme was bound (protected) by the reversible inhibitor in vivo. 

Methods based on the inhibitory effect of the analyte and the use of an enzyme thermistor have primarily been applied to environmental samples and typically involve measuring the inhibitory effect of a pollutant on an enzyme or on the metabolism of appropriate cells [162]. The inhibiting effect of urease was used to develop methods for the determination of heavy metals such as Hg(II), Cu(II) and Ag(I) by use of the enzyme immobilized on CPG. For this purpose, the response obtained for a 0.5-mL standard pulse of urea in phosphate buffer at a flow-rate of 1 mL/min was recorded, after which 0.5 mL of sample was injected. A new 0.5-mL pulse of urea was injected 30 s after the sample pulse (accurate timing was essential) and the response compared with that of the non-inhibited peak. After a sample was run, the initial response could be restored by washing the column with 0.1-0.3 M Nal plus 50 mM EDTA for 3 min. Under these conditions, 50% inhibition (half the initial response) was obtained for a 0.5-mL pulse of 0.04-0.05 mM Hg(II) or Ag(I), or 0.3 mM Cu(II). In some cases, the enzyme was inhibited irreversibly. In this situation, a reversible enzyme immobilization technique... 

The antibacterial and antiviral properties of mono-, di-, and trifluoroalanines are connected to their capacity to inhibit, in an irreversible way, many enzymes (cf. Chapter 1) They have been the subject of numerous synthetic studies. We only report on the most recent and significant ones, without detailing the general methods of amino acid chemistry or the techniques of electrophilic fluorination, which have recently been reviewed. ... 

Acetylcholinesterase inhibition was first performed with the enzyme in solution and detected by colorimetric methods. As the inhibition is irreversible and stoichiometric, it is possible to calculate the toxin concentration by the percentage of the enzyme that has not been inhibited and remains active. The use of several enzyme concentrations and the... 

A number of methods to predict in vivo inhibition from quasi-irreversible or irreversible inhibition have been reported, including the following equation which relates changes in substrate AUC (AUQ-to-AUCui ratio) to the fraction of dose metabolized by the inhibited enzyme ... 

The irreversible inhibition, Kt, of an ICE/ced-3 family protease enzyme using selected experimental agents was determined according to the methods of Thornberry (1) and... 

In conclusion, suicide enzyme inactivators offer a powerful method for the selective irreversible inhibition of enzymes. Although this review has concentrated on pyridoxal phosphate-dependent enzymes the approach is also valid for the irreversible inhibition of other types of enzymes (6 ) and may offer a means for the rational design of therapeutically-useful substances. 

Exploration of a method of finding the active site of an enzyme by irreversible inhibition. 

Unlike the nonspecific effects and uncommon occurrence of direct mortality observed in wildlife exposed to chlorinated hydrocarbon pesticides, several studies have documented direct mortality from exposure to OP and carbamate insecticides. The method by which the OPs and carbamate insecticides affect wildlife is quite different from the method by which the chlorinated hydrocarbon insecticides effect wildlife. The OPs and carbamates inhibit cholinesterase, primarily acetylcholinesterase (AChE), which is an enzyme that functions in the breakdown of the neurotransmitter acetylcholine. Acetylcholine functions in the transmission of nerve impulses. Therefore, when AChE is inhibited by an OP or carbamate insecticide, it can no longer breakdown acetylcholine and there is continued transmission of nerve impulses that eventually leads to nerve and muscle exhaustion. The respiratory muscles are a critical muscle group that is affected, often leading to respiratory paralysis as the immediate cause of death. A major difference in the mode of action between OPs and carbamates is that the inhibition of AChE by OPs is, from a biological standpoint, irreversible, while the inhibition from exposure to carbamates is reversible in a biologically relevant time frame. There... 

Inhibitors are classified into reversible and irreversible types. Reversible inhibition implies that the activity of the enzyme is fiiUy restored when the inhibitor is removed from the system in which the enzyme acts by some physical separative process, such as dialysis, gel filtration, or chromatography. An irreversible inhibitor combines covalently with the enzyme so that physical methods are ineffective in separating the two. For example, organophosphorus compounds are... 

Most biosensors based on AChE have the enzymes immobilized on the surface of the sensor. The inhibition reaction being irreversible, the membrane with immobilized enzyme has to be replaced after several measurements or the biosensor can be use for only one determination. Due to this fact, the researchers tried to realize pesticide biosensors with a renewable surface or disposable biosensors based on screen-printed electrodes (SPE). The screen-printing technology provides a simple, fast and inexpensive method for mass production of disposable biosensors for different biomolecules starting with glucose, lactate and finishing with environmental contaminants as pesticides (Kulys et al., 1991) and herbicides (Skladal, 1992). 

The main value of product inhibition studies of dehydrogenases has been to distinguish between ordered and random mechanisms and to provide additional kinetic estimates of the dissociation constants of enzyme-coenzyme compounds. On both counts the method has been especially useful for reactions that are essentially irreversible or for other reasons cannot be studied in both directions 122,138). It is also in such circumstances that product inhibition studies are most reliable because, as Alberty (7) emphasized when proposing the method, with readily reversible reactions it may be difficult to estimate true initial rates with small concentrations of substrates in the presence of a product. The reality of ternary complexes in an ordered mechanism of the Theorell-Chance type has also been demonstrated with several enzymes (134) by product inhibition studies. 

The previous sections may give the impression that it is an easy matter to establish the mechanism of a multi-substrate enzyme. In fact, more often than not uncertainty and controversy surround such mechanisms for many years despite an abundance of experimental work. We have assumed an ideal situation whereas there are a number of possible obstacles in practice. For example the reaction may be effectively irreversible so that it is only possible to measure the kinetic parameters for one direction of reaction the substrate specificity may be so stringent that it is impossible to apply tests which rely on using a range of alternative substrates the available methods of rate measurement may not be sufficiently sensitive to allow all the kinetic parameters to be determined rehably. The last problem at least is one that allows some hope the kinetic study of NAD-dependent dehydrogenases became much more incisive once fluorescence measurement took over from absorbance measurement as the method of choice [52,57]. Nevertheless there is clearly a need for as many criteria of mechanism as may be mustered and the study of inhibition patterns is a valuable adjunct to the methods already discussed. 

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