Enzyme chemical inhibition

Enzymes can be used not only for the determination of substrates but also for the analysis of enzyme inhibitors. In this type of sensors the response of the detectable species will decrease in the presence of the analyte. The inhibitor may affect the vmax or KM values. Competitive inhibitors, which bind to the same active site than the substrate, will increase the KM value, reflected by a change on the slope of the Lineweaver-Burke plot but will not change vmax. Non-competitive inhibitors, i.e. those that bind to another site of the protein, do not affect KM but produce a decrease in vmax. For instance, the acetylcholinesterase enzyme is inhibited by carbamate and organophosphate pesticides and has been widely used for the development of optical fiber sensors for these compounds based on different chemical transduction schemes (hydrolysis of a colored substrate, pH changes). 

Efforts to overcome the actions of the p-lactamases have led to the development of such p-lactamase inhibitors as clavulanic acid, sulbactam, and tazobactam. They are called suicide inhibitors because they permanently bind when they inactivate p-lactamases. Among the p-lactamase inhibitors, only clavulanic acid is available for oral use. Chemical inhibition of p-lactamases, however, is not a permanent solution to antibiotic resistance, since some p-lactamases are resistant to clavulanic acid, tazobactam, or sulbactam. Enzymes resistant to clavulanic acid include the cephalosporinases produced by Citrobacter spp., Enterobacter spp., and Pseudomonas aeruginosa. 

Acetylsalicyclic acid, aspirin, inhibits the cyclooxygenase-catalyzed first step in the biosynthesis of prostaglandins, prostacyclins and thromboxanes. These latter substances are responsible for the inflammatory and pyretic effects of infection. It is believed that the chemical inhibition reaction involves the acetylation of the enzyme by the aspirin. 

Identification of the P450 form(s) responsible for the metabolism of a xenobiotic can be achieved by correlation of the rates of metabolism of the new entity with the rates of metabolism for marker substrates for specific enzymes in different liver specimens with the levels of the different P450 forms (for review see Wrighton et al., 1993b). A typical approach involves the selection of multiple human tissue preparations with high , medium and low levels of the enzymes of interest. The rate of metabolism of the new entity is assessed in these samples and correlation analyses are performed versus the different marker activities. The enzyme with the highest correlation with the metabolism of the new entity is likely to be the principal enzyme involved in its metabolism. Alternatively, or in addition to correlation analyses, the roles of specific enzymes can be analysed by selective immunoinhibition or chemical inhibition of different P450 forms. The use of chemical inhibitors has been reviewed recently (Halpert et al.,... 

Chemical Inhibition. A large variety of chemical compounds have been added to milk or purified lipase. The conditions under which the inhibitor is studied are very important. Factors such as pH, temperature, time of addition of the chemical, sequence of addition of reactants, and the presence or absence of substrate are undoubtedly involved. The presence of substrate appears to offer some degree of protection to the enzymes. Consequently, in lipase studies, the surface area of the emulsified substrate is probably also important. 

PLP-dependent enzymes are inhibited by a great variety of enzyme-activated inhibitors that react by several distinctly different chemical mechanisms.11 Here are a few. The naturally occurring gabaculline mimics y-aminobutyrate (Gaba) and inhibits y-aminobutyrate aminotransferase as well as other PLP-dependent enzymes. The inhibitor follows the normal catalytic pathway as far as the ketimine. There, a proton is lost from the inhibitor permitting formation of a stable benzene ring and leaving the inhibitor stuck in the active site ... 

Chemical inhibition, which involves an evaluation of the effects of known CYP enzyme inhibitors on the metabolism of a drug candidate by human liver microsomes. Chemical inhibitors of CYP must be used cautiously because most of them can inhibit more than one CYP enzyme and some chemicals can inhibit one enzyme but activate another. Some chemical inhibitors are mechanism-based inhibitors that require biotransformation to a metabolite that inhibits or inactivates CYP. 

The FDA-approved and acceptable chemical inhibitors for reaction phenotyping are included in Table 2. Many of the inhibitors listed in Table 2 are metabolism-dependent inhibitors that, in order to inhibit CYP, require preincubation with NADPH-fortified human liver microsomes for 15 minutes or more. In the absence of the metabolism-dependent inhibitor, this preincubation of microsomes with NADPH can result in the partial, spontaneous loss of several CYP enzyme activities (see sec. II.C.7.c). Furthermore, the organic solvents commonly used to dissolve chemical inhibitors can themselves inhibit (or possibly activate) certain CYP enzymes, as discussed in section II.C.4. Therefore, appropriate solvent and preincubation controls should be included in all chemical inhibition experiments. 

As in the case of chemical inhibition, a lack of specificity can complicate the interpretation of antibody inhibition experiments. A lack of specificity and the nonspecific effects outlined above likely account for the majority of cases where the sum of the inhibitory effects of a panel of inhibitory antibodies adds up to greater than 100%. Another potential problem stems from the fact that many antibodies do not completely inhibit the activity of a microsomal CYP enzyme or the corresponding recombinant CYP enzyme. If an antibody inhibits the metabolism of a marker substrate by 80%, and if the same antibody inhibits the metabolism of drug candidate by 80%, there is uncertainty as to whether the inhibited enzyme contributes 80% or 100% to the metabolism of the drug candidate. This may seem like a trivial difference, but it has a... 

The FDA guidance document recognizes the difficulty of extrapolating the results obtained with recombinant enzymes to the situation in liver microsomes. Experiments with recombinant CYP enzymes provide valuable information on which CYP enzymes can and which ones cannot convert a drug candidate to a particular metabolite, and this information alone is particularly valuable in guiding the design or interpretation of correlation analysis, chemical inhibition, and antibody inhibition experiments. 

The biochemical reaction performed by the methyltransferase appears to be metal dependent, as determined by chemical inhibition studies, but the exact identity of this metal has yet to be defined [41]. EDTA treatment had no inhibitory effect on Stel4p, but incubation of the protein with 10 - 50 mM of the metal chelating agent 1,10-phenanthroline eliminated catalytic activity [37,41]. Additionally, even more hydrophobic metal sequesterants such as zincon [37], lysine nitriloacetic acid (Lys-NTA) [42], and cholesteryl-Lys-NTA [43] also inhibited the methyltransferase but at much lower concentrations. Together, these data suggest that the metal ion may be buried in a hydrophobic region of the enzyme. 

The structure-activity relationship of HVK -ATPase inhibitors of the omeprazole type is based on the balance between chemical stability at neutral pH values and acid-induced conversion into the active sulphenamide. Derivatives, which are too unstable at neutral pH, are very active in the test assay of partly purified HVK" -ATPase. This assay has been performed at pH 7.4 after preincubation at pH 6 of the enzyme protein with the derivative to be tested. The high activity was therefore the result of the conversion of the derivative in solutions of neutral pH values and this does not reflect the situation of high acidity within the secretory compartment of the parietal cell [28]. The derivatives which are very unstable at neutral pH do not inhibit gastric acid secretion in vivo because their transformation had already occurred prior to the active principle reaching the target enzyme. Chemically very stable derivatives do not show any inhibitory effect either in vitro or in vivo. 

Competitive product inhibition and allosteric regulation (fastest). Many enzymes are inhibited by either their products, or by other chemicals, often those from further down a metabolic pathway. Such enzymes may be gatekeepers to a specific branch of metabolism, and they usually catalyse a true equilibrium reaction, j.e., one that doesn t go to completion (note this is not exactly the same reaction in the forward and backwards directions, so we are not defying the law that states enzymes do not alter the equilibrium point). 

Takaoka, Y, Kajimoto, T, Wong, C-H, Inhibition of iV-acetyl-glucosaminyltransfer enzymes chemical-enzymatic synthesis of new five-membered acetamido azasugars, J. Org. Chem., 58, 4809-4812, 1993. 

PLP-dependent enzymes are inhibited by a great variety of enzyme-activated inhibitors that react by several distinctly different chemical mechanisms. ... 

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