Experiments, enzyme inhibition

Because of some of the problems with bioassays and immunoassays, liquid chromatography (LC)-based techniques are increasingly applied as an alternative. While modern LC-based assays have a comparable sensitivity to immunoassays, they oftentimes are characterized by a higher selectivity [18, 19]. Muller et ah, for example, used LC/mass spectrometry with matrix-assisted laser desorption ionization in ex vivo pharmacokinetic studies in combination with enzyme inhibition experiments to investigate the complex metabolism of dynorphin Al-13, a peptide with opioid activity, up to the fifth metabolite generation [20, 21]. 

Somatostatin is a tetradecapeptide of the hypothalamus that inhibits the release of pituitary growth hormone. Its amino acid sequence has been determined by a combination of Edman degradations and enzymic hydrolysis experiments. On the basis of the following data, deduce the primary structure of somatostatin ... 

Inhibition experiments are performed by varying the concentration of substrate around the Km just as you would in an experiment to determine the Km and Vmax, except that the experiment is repeated at several different concentrations of an inhibitor. On a Lineweaver-Burk transformation (1/v vs. 1/[S]), each different inhibitor concentration will be represented as a different straight line (Fig. 8-6). The pattern that the lines make tells you the kind of inhibition. There are three possibilities (1) The inhibitor can affect only the slopes of the plot (competitive), (2) the inhibitor can affect only the y intercepts of the plot (uncompetitive), or (3) the inhibitor can affect both the slopes and the intercepts (noncompetitive). Plots are plots, and what s really important is not the pattern on a piece of paper but what the pattern tells us about the behavior of the enzyme. 

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. 

It was suggested,1 on the basis of kinetic measurements, that the phosphorofluoridates inhibit esterases by virtue of a highly specific affinity for the active centres of this group of enzymes. Preliminary experiments by Boursnell and Webb2 with diisopropyl phosphorofluoridate containing 32P gave results which were in accordance with this view. 

This linearization of the tight-binding scheme allows the investigator the opportunity to calculate values for [Etotai] and Ki, the dissociation constant for the inhibitor. In the Henderson plot, [Itotai]/(l v/Vo) is plotted as a function of vjv where Vq is the steady-state velocity of the reaction in the absence of the inhibitor. The slope of the line is the apparent dissociation constant for the inhibitor. Secondary plots (from repeating the inhibition experiment at different substrate concentrations) will yield the Ki value. The vertical intercept is equal to [Etotai]- Hence, repeating the experiment at a different concentration of enzyme will produce a parallel line. 

Fromm and Rudolph have discussed the practical limitations on interpreting product inhibition experiments. The table below illustrates the distinctive kinetic patterns observed with bisubstrate enzymes in the absence or presence of abortive complex formation. It should also be noted that the random mechanisms in this table (and in similar tables in other texts) are usually for rapid equilibrium random mechanism schemes. Steady-state random mechanisms will contain squared terms in the product concentrations in the overall rate expression. The presence of these terms would predict nonhnearity in product inhibition studies. This nonlin-earity might not be obvious under standard initial rate protocols, but products that would be competitive in rapid equilibrium systems might appear to be noncompetitive in steady-state random schemes , depending on the relative magnitude of those squared terms. See Abortive Complex... 

The whole question of the specificity was reopened with the discovery that E. coli phosphatase, contrary to an earlier statement (114), hydrolyzed a variety of polyphosphates including metaphosphate of average chain length 8 (97). It was subsequently reported that partially purified phosphatases from several mammalian tissues had appreciable PPi-ase activity at pH 8.5 (115). This was confirmed (116) and extended to include ATPase and fluorophosphatase activities (117). Proof that the same enzyme is responsible for the monoesterase and PPi-ase activities was afforded by heat inactivation studies, cross inhibition experiments, and inhibition of PPi-ase activity by L-phenylalanine, a specific inhibitor of intestinal phosphatase. It was also found that calf intestinal phosphatase couid be phosphorylated by 32P-PP and the number of sites so labeled agreed with the number of active sites determined with a monoester substrate using a stopped-flow technique (118). It would seem that the main reason for the confusion with regard to the PPi-ase activity results from the inclusion of Mg2+ in the assay. This stimulates the monoesterase activity but almost completely inhibits PPi-ase activity (117). 

First hints on the nature of regulatory sites in yeast PDC have been obtained from inhibition experiments with the thiol-modifying reagents 4-hydroxy-mer-curi-benzoate and 3-bromo-pyruvamide in the presence and absence of the substrate pyruvate. Upon thiol modification, PDC lost its activation behaviour. Pyruvate was able to protect the enzyme against thiol-modification, suggesting that both modifier and substrate compete for the same sites on the enzyme... 

Sortino et al. reported the use of light irradiation on SAMs supporting the anticancer drug flutamide to study its photoreactivity and product release of nitric oxide. The authors were able to conduct studies in the absence of noxious side effects such as singlet-oxygen photosensitization and observed that nitric oxide production halted when the light was turned off [103], Another photoactive molecule, azobenzene, has recently been exploited in the SAM community to aid in studies on cell adhesion and enzyme inhibition [104], Pearson and coworkers described the use of photoisomerizable SAMs of azobenzene to conduct a series of serine and cysteine protease inhibition experiments. When light of 340-380 nm is used, the... 

Solvents should be kept to <1% (v/v) and preferably <0.1% (v/v) because some solvents inhibit one or more CYP enzymes. The experiment may include a no-solvent control as well as a solvent control to determine the effects of the solvent. 

Chemical and antibody inhibition represent the second and third approaches to reaction phenotyping. They typically involve an evaluation of the effects of known CYP enzyme inhibitors or inhibitory antibodies against selected CYP enzymes on the metabolism of a drug candidate by pooled human liver micro-somes. As in the case of correlation analysis, chemical and antibody inhibition experiments must be conducted with pharmacologically relevant concentrations of the drug candidate in order to obtain clinically relevant results. 

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. 

---