Enzymatic experimental methods

For the quantitative description of the metabolic state of a cell, and likewise which is of particular interest within this review as input for metabolic models, experimental information about the level of metabolites is pivotal. Over the last decades, a variety of experimental methods for metabolite quantification have been developed, each with specific scopes and limits. While some methods aim at an exact quantification of single metabolites, other methods aim to capture relative levels of as many metabolites as possible. However, before providing an overview about the different methods for metabolite measurements, it is essential to recall that the time scales of metabolism are very fast Accordingly, for invasive methods samples have to be taken quickly and metabolism has to be stopped, usually by quick-freezing, for example, in liquid nitrogen. Subsequently, all further processing has to be performed in a way that prevents enzymatic reactions to proceed, either by separating enzymes and metabolites or by suspension in a nonpolar solvent. 

Depending on the probe drug used and on the experimental method, 2 or 3 acetylator types can be described slow, intermediate and rapid, the intermediate one being not always distinguished from the rapid one. Phenotype distribution has been considered as a continuous variable (Meisel 2002). Due to slow post-natal maturation of the acetylation enzymatic systems, the acetylation status is evolving in newborns and infants, and depends on the probe drug used (Rane 1999). 

The quantitation of enzymes and substrates has long been of critical importance in clinical chemistry, since metabolic levels of a variety of species are known to be associated with certain disease states. Enzymatic methods may be used in complex matrices, such as serum or urine, due to the high selectivity of enzymes for their natural substrates. Because of this selectivity, enzymatic assays are also used in chemical and biochemical research. This chapter considers quantitative experimental methods, the biochemical species that is being measured, how the measurement is made, and how experimental data relate to concentration. This chapter assumes familiarity with the principles of spectroscopic (absorbance, fluorescence, chemi-and bioluminescence, nephelometry, and turbidimetry), electrochemical (poten-tiometry and amperometry), calorimetry, and radiochemical methods. For an excellent coverage of these topics, the student is referred to Daniel C. Harris, Quantitative Chemical Analysis (6th ed.). In addition, statistical terms and methods, such as detection limit, signal-to-noise ratio (S/N), sensitivity, relative standard deviation (RSD), and linear regression are assumed familiar Chapter 16 in this volume discusses statistical parameters. 

Amino acid surfactants (AAS), both natural and synthetic types, have been the subject of many smdies, due mostly to their huge potential application in pharmaceutical, cosmetic, household, and food products. The AAS are derived from acidic, basic, or neutral amino acids. Amino acids such as glutamic acid, glycine, alanine, arginine, aspartic acid, leucine, serine, proline, and protein hydrolysates have been used as starting materials to synthesize AAS commercially and experimentally. Methods of preparation include chemical, enzymatic, and chemoenzymic processes, although chemical processes have been prevalent due to their relatively low cost of production. In recent years, more research papers have focused on the use of enzymatic methods to synthesize AAS. It is our opinion that the enzymatic approach would be more attractive to manufacturers in the near future. 

It seems to us that the complicated interrelations between structure and function in enzyme catalysis cannot be fully understood without a model that takes all the relevant interactions into account. If one can devise sufficiently accurate schemes for simulating enzymatic reactions and reproducing the observed rate constants, it would be possible to examine the different contributions to the calculated activation energy and evaluate their relative importance. This would also make it possible to explore the detailed mechanisms of enzyme catalysis in a way that is not accessible to direct experimental methods (e.g. with a reliable computational simulation scheme, the relative importance of such factors as strain and electrostatics can be readily evaluated). However, it is, important to realise that in order for a theoretical framework to be really useful in this context, it should be able to give semi-quantitative or quantitative information, rather than just providing an exercise in computational quantum chemistry at the qualitative level. 

The plotting of Dixon plot and its slope re-plot (see 5.9.5.9) is a commonly used graphical method for verification of kinetics mechanisms in a particular enzymatic reaction.9 The proposed kinetic mechanism for the system is valid if the experimental data fit the rate equation given by (5.9.4.4). In this attempt, different sets of experimental data for kinetic resolution of racemic ibuprofen ester by immobilised lipase in EMR were fitted into the rate equation of (5.7.5.6). The Dixon plot is presented in Figure 5.22. 

The experimental evidences that medium engineering might represent an efficient method to modify or improve enzyme selectivity (alternative to protein engineering and to the time-consuming search for new catalysts) were immediately matched by the search for a sound rationale of this phenomenon. The different hypotheses formulated to try to rationalize the effects of the solvent on enzymatic enantioselectivity can be grouped into three different classes. The first hypothesis suggests that... 

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

Today a good understanding of transition state structure can be obtained through a combination of experimental measurements of kinetic isotope effects (KIE) and computational chemistry methods (Schramm, 1998). The basis for the KIE approach is that incorporation of a heavy isotope, at a specific atom in a substrate molecule, will affect the enzymatic reaction rate to an extent that is correlated with the change in bond vibrational environment for that atom, in going from the ground state to the... 

The experimental objective of the study was to obtain a series of stop-action photographs of ribonuclease A at work at atomic resolution. The strategy for such a program has been considered in detail by Fink and Petsko (1981), who treat such subjects as diffusional constraints and turnover rates, and in the preceding sections of this article. The ribonuclease reaction has a series of well-characterized, stable species which can be purchased, and crystals of the enzyme are large, well ordered, catalyt-ically active (Fink et al, 1984), and have as their natural mother liquor a cryoprotective solvent (Petsko, 1975). RNase thus represents the ideal system for a step-by-step analysis of an enzymatic catalytic pathway by the methods outlined above. 

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