Reversible enzyme inhibition method

Because of the complexity of biological systems, Eq. (1) as the differential form of Michaelis-Menten kinetics is often analyzed using the initial rate method. Due to the restriction of the initial range of conversion, unwanted influences such as reversible product formation, effects due to enzyme inhibition, or side reactions are reduced to a minimum. The major disadvantage of this procedure is that a relatively large number of experiments must be conducted in order to determine the desired rate constants. 

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 simplest method to determine whether an inhibitor is reversible after enzyme inhibition with excess inhibitor is to remove the inhibitor by dialysis or gel filtration. If full enzyme activity returns, then inhibition is reversible. However, it... 

Guidelines issued by JMPR have drawn attention to the currently used methods for the determination of ChE activity that may lead to erroneous conclusions when applied to rapidly reversible ChE inhibitions (c.g., JV-methyl and N,N-dimethyl CMs). It has been suggested that in vitro kinetic studies should be made to elucidate the nature of reversible inhibition reactions and the results obtained from in vivo studies should be interpreted cautiously. The method for the determination of ChE inhibition of CMs i.s inadequate. Occasionally, data are inconsistent with respect to dose and the degree of ChE inhibition because CMs are reversible inhibitors of ChE with a short duration of action. Because of the reversible inhibition of the enzyme by dilution, as would occur during the preparation of the assay, inhibition cannot be accurately measured. JMPR has stressed that in order to permit evaluation of ChE inhibition by CMs in viva, special care is required in reporting all details of such studies. CM-induced ChE inhibition studies should utilize minimal dilution during the preparation of the assay, minimal incubation limes, and minimal times between blood sampling and assay (Ellman etui, 1967),... 

If the inhibitor is found to bind rapidly (linear progress curves) and dissociate rapidly (rapid recovery of activity upon dilution) from its target enzyme, then one can proceed to analyze its inhibition modality and affinity by classical methods. The modes of reversible inhibition of enzymes were described in Chapter 3. In the next section of this chapter we will describe convenient methods for determining reversible inhibition modality of lead compounds and lead analogues during compound optimization (i.e., SAR) studies. 

In AChE-based biosensors acetylthiocholine is commonly used as a substrate. The thiocholine produced during the catalytic reaction can be monitored using spectromet-ric, amperometric [44] (Fig. 2.2) or potentiometric methods. The enzyme activity is indirectly proportional to the pesticide concentration. La Rosa et al. [45] used 4-ami-nophenyl acetate as the enzyme substrate for a cholinesterase sensor for pesticide determination. This system allowed the determination of esterase activities via oxidation of the enzymatic product 4-aminophenol rather than the typical thiocholine. Sulfonylureas are reversible inhibitors of acetolactate synthase (ALS). By taking advantage of this inhibition mechanism ALS has been entrapped in photo cured polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ) to prepare an amperometric biosensor for... 

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. 

Selected entries from Methods in Enzymology [vol, page(s)] Theory, 63, 159-162 activation effect, 63, 174, 175 analysis, 63, 140, 159-183 burst, 64, 20, 203, 215 enzyme concentration, 63, 175-177 hysteresis, 64, 197, 200-204 limitations, 63, 181-183 plotting, 63, 177-180 practical methods, 63, 175-177 reversible inhibitor action, 63, 163-175 reversible reaction, 63, 171-175 simulation of, 63, 180 advantages and disadvantages, 249, 61-62 analysis, in kinetic models of inhibition, 249, 168-169 concave-down, 249, 156 concave-up, 249, 156 with enzyme-product complex instability, 249, 88 with enzyme-substrate instabil-... 

The majority of preparations in clinical phases have been found through conventional screening methods and interfere in the inhibition of either reverse transcriptase or HIV protease. The cycle of replication is shown in Figure 13.13. New programs target control and inhibition of all three HIV enzymes, such as reverse transcriptase, protease, and integrase, as well as regulator proteins such as Tat,... 

Figure 10.4 Examples of covalent capture methods. (A) Covalent modification of native cysteines has been shown to modulate ion-channel activity and in the case of enzymes lead to allosteric inhibition. These findings can be important for structural-functional characterization or as starting points for drug discovery. (B) Reversible covalent capture using imine chemistry.

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