Enzymes quantitative data

The great power of mechanistic enzymology in drug discovery is the quantitative nature of the information gleaned from these studies, and the direct utility of this quantitative data in driving compound optimization. For this reason any meaningful description of enzyme-inhibitor interactions must rest on a solid mathematical foundation. Thus, where appropriate, mathematical formulas are presented in each chapter to help the reader understand the concepts and the correct evaluation of the experimental data. To the extent possible, however, I have tried to keep the mathematics to a minimum, and instead have attempted to provide more descriptive accounts of the molecular interactions that drive enzyme-inhibitor interactions. 

While there have been a considerable number of structural models for these multinuclear zinc enzymes (49), there have only been a few functional models until now. Czamik et al. have reported phosphate hydrolysis with bis(Coni-cyclen) complexes 39 (50) and 40 (51). The flexible binuclear cobalt(III) complex 39 (1 mM) hydrolyzed bis(4-nitro-phenyl)phosphate (BNP-) (0.05 mM) at pH 7 and 25°C with a rate 3.2 times faster than the parent Coni-cyclen (2 mM). The more rigid complex 40 was designed to accommodate inorganic phosphate in the in-temuclear pocket and to prevent formation of an intramolecular ju.-oxo dinuclear complex. The dinuclear cobalt(III) complex 40 (1 mM) indeed hydrolyzed 4-nitrophenyl phosphate (NP2-) (0.025 mM) 10 times faster than Coni-cyclen (2 mM) at pH 7 and 25°C (see Scheme 10). The final product was postulated to be 41 on the basis of 31P NMR analysis. In 40, one cobalt(III) ion probably provides a nucleophilic water molecule, while the second cobalt(III) binds the phosphoryl group in the form of a four-membered ring (see 42). The reaction of the phosphomonoester NP2- can therefore profit from the special placement of the two metal ions. As expected from the weaker interaction of BNP- with cobalt(in), 40 did not show enhanced reactivity toward BNP-. However, in the absence of more quantitative data, a detailed reaction mechanism cannot be drawn. 

There are no adequate data to evaluate whether pharmacokinetics of DEHP in children are different from adults. It is not known whether DEHP (or metabolites) can cross the placenta in humans, although it has been detected in breast milk (FDA 200 lh). Studies in animals have shown that DEHP (or metabolites) crosses the placenta and can be transferred to offspring via mother s milk however, quantitative data are lacking. There is no information to evaluate whether metabolism of DEHP is different in children than in adults since the specific phase I enzymes involved in DEHP metabolism have not been identified. It is known that phase II metabolism involves conjugation with glucuronic acid, but the specific isoform of glucuronosyltransferase is not known. 

Once a suitable concentration of enzyme has been established so that three or four samples can be analyzed, the quantitative data can be obtained. A reaction is started, the reaction mixture is sampled at intervals, chromatograms are obtained, and the amount of product formed is determined directly from the chromatogram by means of either peak height or electronic integration of the peaks. These values should be plotted as a function of reaction time. 

In many clinical laboratories, gel-based methods are restricted to qualitative studies. Quantitative data are more commonly obtained by turbidimetric and nephelometric methods, radioimmunoassays, enzyme immunoassays (EIAs), and fluorometric immunoassays. 

Another procedure involves enzymatic hydrolysis of monoterpene glycoside mixtures to liberate monoterpenes which can then be analyzed by gas chromatography (26,34). Such methods provide identifications and give good quantitative data for the important aglycon, but not the sugar moieties. When polyols are released by the enzyme, no evidence can be obtained about the site of attachment of the carbohydrates to the terpenols. 

Epithelial cells migrate from the crypts to the tips of the villi in about 2 days [11]. During this time they differentiate and, at the end of their life cycle, are shed into the lumen. Their contents are extruded in the succus entericus and the intracellular enzymes become available for food and drug metabolization in the gut. The amount of cellular material liberated is about 250 g per day. Drugs and macromolecules can be absorbed by this first paracellular pathway, but no quantitative data are as yet available concerning the extent of this phenomenon. Recent literature suggests that some authors are in favor of absorption between the cells, called persorption and responsible for the uptake of cells [15], while others are still skeptical. 

Because of its easiness, in several studies a so-called in vivo enzyme activity assay was performed. This assay uses the amounts and also the composition of PHAs that are synthesized per time by whole cells during their cultivation as an indication for enzyme activity and PHA substrate specificity, respectively. Although such an assay allows the demonstration of the functionality of the PhaC protein, quantitative data can hardly be obtained because the conditions of the PhaC with regard to substrate concentration and the presence of other biological molecules, which affect the enzyme activity in a positive or negative way, can hardly be controlled. 

One major advantage of the glow-type emission obtained from dioxetane enzyme substrates is that one can easily record the emission on photographic film (in much the same way that emission is recorded) to obtain qualitative or semi-quantitative data. This technique has been described for a dry-format membrane-based assay for human luteinizing hormone (hLH), in which Polaroid 612 film (ASA 20,000) was used to record the intensity from unknown samples and from a set of standards (B18). A dioxetane-based ELISA method for human growth hormone (somatotropin) has been recently described. The chemiluminescent assay was reported to be superior to a similar fluorescence assay, and the detection limit for hGH was found to be 5 pM (A9). 

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