Properties of Enzyme Catalyzed Reactions

Enzymes are complex proteins that act as catalysts in various biochemical interactions. They generally function at ambient temperatures, are specific in action, have an optimum pH, and are effective at very low concentrations. A brief discussion of some of the general properties of enzyme catalyzed reactions follows below. 

FIGURE 9.1 The influence of temperature on reaction rate of a typical enzyme catalyzed reaction. (From Mathewson, P.R. Enzymes, Eagan Press, St. Paul, 2004. With permission.) 

FIGURE 9.3 The effect of shear on the inactivation of a typical enzyme. (From Richardson, T., D.B. Hyslop, Food Chemistry, 2nd ed., O.R. Fennema, Ed., Dekker, New York. With 

---

Calculations of Standard Transformed Thermodynamic Properties of Enzyme-catalyzed Reactions... 

This package provides data on the species of 131 reactants at 298.15 K and programs for calculating various transformed thermodynamic properties. Programs are given for the calculation of apparent equilibrium constants and other transformed thermodynamic properties of enzyme-catalyzed reactions by simply typing in the reaction. 

Immobilization. The fixing property of PEIs has previously been discussed. Another appHcation of this property is enzyme immobilization (419). Enzymes can be bound by reactive compounds, eg, isothiocyanate (420) to the PEI skeleton, or immobilized on soHd supports, eg, cotton by adhesion with the aid of PEIs. In every case, fixing considerably simplifies the performance of enzyme-catalyzed reactions, thus faciHtating preparative work. This technique has been appHed to glutaraldehyde-sensitive enzymes (421), a-glucose transferase (422), and pectin lyase, pectin esterase, and endopolygalacturonase (423). 

In this chapter, a short introduction to DFT and to its implementation in the so-called ab initio molecular dynamics (AIMD) method will be given first. Then, focusing mainly on our own work, applications of DFT to such fields as the definition of structure-activity relationships (SAR) of bioactive compounds, the interpretation of the mechanism of enzyme-catalyzed reactions, and the study of the physicochemical properties of transition metal complexes will be reviewed. Where possible, a case study will be examined, and other applications will be described in less detail. 

To make QM studies of chemical reactions in the condensed phase computationally more feasible combined quantum me-chanical/molecular mechanical (QM/MM) methods have been developed. The idea of combined QM/MM methods, introduced first by Levitt and Warshell [17] in 1976, is to divide the system into a part which is treated accurately by means of quantum mechanics and a part whose properties are approximated by use of QM methods (Fig. 5.1). Typically, QM methods are used to describe chemical processes in which bonds are broken and formed, or electron-transfer and excitation processes, which cannot be treated with MM methods. Combined QM and MM methods have been extensively used to study chemical reactions in solution and the mechanisms of enzyme-catalyzed reactions. When the system is partitioned into the QM and MM parts it is assumed that the process requiring QM treatment is localized in that region. The MM methods are then used to approximate the effects of the environment on the QM part of the system, which, via steric and electrostatic interactions, can be substantial. The... 

The kinetics of enzyme-catalyzed reactions (i. e the dependence of the reaction rate on the reaction conditions) is mainly determined by the properties of the catalyst, it is therefore more complex than the kinetics of an uncatalyzed reaction (see p.22). Here we discuss these issues using the example of a simple first-order reaction (see p.22)... 

The procedure for calculating standard formation properties of species at zero ionic strength from measurements of apparent equilibrium constants is discussed in the next chapter. The future of the thermodynamics of species in aqueous solutions depends largely on the use of enzyme-catalyzed reactions. The reason that more complicated ions in aqueous solutions were not included in the NBS Tables (1992) is that it is difficult to determine equilibrium constants in systems where a number of reactions occur simultaneously. Since many enzymes catalyze clean-cut reactions, they make it possible to determine apparent equilibrium constants and heats of reaction between very complicated organic reactants that could not have been studied classically. 

The semigrand partition function F corresponds with a system of enzyme-catalyzed reactions in contact with a reservoir of hydrogen ions at a specified pH. The semigrand partition function can be written for an aqueous solution of a biochemical reactant at specified pH or a system involving many biochemical reations. The other thermodynamic properties of the system can be calculated from F. 

Several thermodynamic and kinetic behaviors of enzyme-catalyzed reactions performed in ILs, with respect to enzymatic reactions carried out in conventional solvents, could lead to an improvement in the process performance [34—37]. ILs showed an over-stabilization effect on biocatalysts [38] on the basis of the double role played by these neoteric solvents ILs could provide an adequate microenvironment for the catalytic action of the enzyme (mass transfer phenomena and active catalytic conformation) and if they act as a solvent, ILs may be regarded as liquid immobilization supports, since multipoint enzyme-1L interactions (hydrogen. Van der Waals, ionic, etc.) may occur, resulting in a flexible supramolecular not able to maintain the active protein conformation [39]. Their polar and non-coordinating properties hold considerable potential for enantioselective reactions since profound effects on reactivities and selectivities are expected [40]. In recent years attention has been focused on the appUcation of ILs as reaction media for enantioselective processes [41—43]. 

The activating cation presumably acts by helping the protein to maintain a productive conformation, and examples are provided by structural studies of enzymes that exhibit this property. Pauling suggested that complementarity between the stracture of the enzyme active site and the transition state of the reaction is responsible for the lowering of activation energies of enzyme-catalyzed reactions. Cation activation may aid this by ensuring that the active site has the correct... 

The functions derived in the preceding section can be added and subtracted to obtain standard transformed thermodynamic properties for enzyme-catalyzed reactions. The program deriveftiGHSNHrx is used to produce a list of functions for the reaction properties A, G A, H A, S °, and Nh for a typed-in reaction that can be used to calculate tables or make... 

An understanding of acid-base chemistry is essential if we are to appreciate the properties of biological molecules. A great many of the low-molecular-weight metabolites and macromolecular components of living cells are acids and bases, and thus, have the potential to ionize. The electrical charges on these molecules are important factors in the rate of enzyme-catalyzed reactions, the stability and conformation of proteins, the interactions of macromolecules with each other and with small ions, and the analytical and purification techniques used in the laboratory. 

Since acidity and basicity are chemical properties, their true measure would be their effects on reactions. A large number of enzyme-catalyzed reactions known to be markedly pH-dependent and... 

The first applications of enzymes in bioanalytical chemistry can be dated back to the middle of nineteenth century, and they were also used for design of first biosensors. These enzymes, which have proved particularly useful in development of biosensors, are able to stabilize the transition state between substrate and its products at the active sites. Enzymes are classified regarding their functions, and the classes of enzymes are relevant to different types of biosensors. The increase in reaction rate that occurs in enzyme-catalyzed reactions may range from several up to e.g. 13 orders of magnitude observed for hydrolysis of urea in the presence of urease. Kinetic properties of enzymes are most commonly expressed by Michaelis constant Ku that corresponds to concentration of substrate required to achieve half of the maximum rate of enzyme-catalyzed reaction. When enzyme is saturated, the reaction rate depends only on the turnover number, i.e., number of substrate molecules reacting per second. 

Because of their tunable properties, supercritical solvents provide a useful medium for enzyme-catalyzed reactions.f The mechanism of enzyme-catalyzed reactions is similar to the mechanism described for solid-catalyzed reactions. External as well as internal transport effects may limit the reaction rate. Utilizing supercritical fluids enhances external transport rate due to increase in the diffusivity and therefore mass transfer coefficient. Internal transport rate depends on the fluid medium as well as the morphology of the enzyme. Supercritical fluids can alter both. 

Aspects that are generally treated in texts on food chemistry are for the most part left out an example is the mechanism and kinetics of enzyme-catalyzed reactions. Some subjects are not fully treated, such as rheological and other mechanical properties, since this would take very much space, and several books on the subject exist. 

The kinetics of enzyme-catalyzed reactions can be very complex, and the mathematical representations for the effect of the concentrations of substrate, product, cofactors, and inhibitors are presented in a variety of textbooks in this field [1]. The exact form of this dependence of enzyme activity on these factors might have a profound effect on the behavior of an enzyme biosensor. However, one can delineate general rules of thumb concerning the properties of enzymes for the preliminary design of enzyme-based sensors. 

Because enzymes are polyionic polymers, it is expected that pH will affect most of their properties. In fact, change in pH may change the distribution of charges in the active site and in the whole surface of the protein molecule. Enzymes may present polar amino acid residues at its active site whose charge depends on pH. With respect to enzyme activity, it is a well known fact that rates of enzyme catalyzed reactions tend to decrease at extremes of pH usually exhibiting maxima at some intermediate values as shown in Fig. 3.10 for the case of glucoamylase (Illanes 1983). 

The rates of enzyme-catalyzed reactions can be followed, not only by a wide range of instrumental techniques, but also with the aid of a variety of chemical principles. In favorable cases, reaction rates can be determined directly by observing consumption of the substrate or appearance of the product, but if these compounds lack distinctive physical properties, indirect ap-proache.s, espiecially coupled reactions, can be used (see Section 9.1.3.2). 

Virtually all enzymatic assays are carried out at 20-50 °C in aqueous buffers of known pH and controlled composition. Both temperature and buffer properties affect the rates of enzyme- catalyzed reactions markedly. The effects of temperature can usually be summarized by a bell-shaped curve (Fig. 4 A). At lower temperatures, reaction rates increase with temperature, but beyond a certain point, denaturation (unfolding) of the enzyme molecules begins, so they lose their ability to bind the substrate, and the reaction rate falls. The temperature giving maximum activity varies from one enzyme to another, according to the robustness of the molecule. In some cases, it may be convenient to use a temperature rather below this maximum, otherwise the rate becomes too high to measure precisely. The rates of many enzyme-catalyzed reactions increase by a factor of ca. 2 over a range of I0°C in the region below the maximum of the... 

The properties of stationary structures of enzymatic processes can be different from those obtained in gas phase calculations because, obviously, the interaction with the environment are not considered in the latter. Then, a more realistic picture of enzyme catalyzed reactions can be obtained including a small part of the active centre into the calculations. The problem is that in this cluster or supermolecule models, the optimised stmetures do not necessary fit into the enzyme active site and computing artefacts can be obtained. A common strategy is to anchor some key atoms of the enzyme to their crystallographic positions and then optimize the rest of the coordinates of the model [39]. Nevertheless, this is an approximate solution that presents several deficiencies. First, the result will be dependent on the initial X-ray stmeture that, in many cases, is far from the real stmeture of the protein-substrate complex at the TS. Second, long-range effects on the nuclear and electronic polarisation of the chemical system are not included in the calculations. Third, and probably the most dramatic deficiency, the enzyme flexibility is not properly taken into account. And finally, the computational cost of these calculations rapidly increases, as more atoms of the environment are explicidy included. 

---