Enzyme factors affecting catalysis

Enzymes use the same catalytic mechanisms as nonenzymatic catalysts. Several factors contribute to enzyme catalysis proximity and strain effects, electrostatic effects, acid-base catalysis, and covalent catalysis. Combinations of these factors affect enzyme mechanisms. 

This chapter first explains enzyme nomenclature, describes enzymatic, supercritical reactor configurations, and gives a compilation of published experimental results. The- most important topics concerning enzymatic reactions in SCFs are then covered. These are factors affecting enzyme stability, the role of water in enzymatic catalysis, and the effect of pressure on reaction rates. Studies on mass transfer effects are also reviewed as are factors that have an effect on reaction selectivities. Finally, a rough cost calculation for a hypothetical industrial process is given. 

Following the classical syntheses of aromatic a-sialosides by phase transfer catalysis starting with the per-O-acetylated sialosyl chloride methyl ester, further aromatic a-sialosides could be prepared with sesamol (4), 2-chloro -nitrophenol (5), and 4-chloro-5-methyl-4-nitrophenol (6) [36]. In TcTS-sialylations none of these modified donor substrates showed any sialylation of methyl p-lactoside (7) to give the methyl sialyllactoside (8). It may be assumed that both steric and electronic factors affect interactions with aromatic and/or hydrophobic amino acid residues in the enzyme (Scheme 1). 

Enzymatic Catalysis. Enzymes are biological catalysts. They increase the rate of a chemical reaction without undergoing permanent change and without affecting the reaction equiUbrium. The thermodynamic approach to the study of a chemical reaction calculates the equiUbrium concentrations using the thermodynamic properties of the substrates and products. This approach gives no information about the rate at which the equiUbrium is reached. The kinetic approach is concerned with the reaction rates and the factors that determine these, eg, pH, temperature, and presence of a catalyst. Therefore, the kinetic approach is essentially an experimental investigation. 

The factors — or at least some of them — which control reactivity in intramolecular reactions are relevant to enzyme catalysis, which also involves reactions between functional groups brought together in close and precisely defined proximity (Kirby, 1980). This has been an area of lively discussion in the recent literature [for a brief summary and leading references see Paquette et al. (1990)]. The main difficulty in making generalizations about the dependence of reactivity on geometry based on results from systems in which proximity is covalently enforced lies in the constraints imposed by particular systems. These may well affect reactivity... 

In the past the mineral matrix was considered as inert, only providing stabilization support for enzymes and humic substances however, due to the overwhelming amount of evidence at the molecular level, there is no doubt that minerals participate in abiotic catalysis of humification reactions in soils. Naidja et al. (2000) referred to mineral particles as the Hidden Half of enzyme-clay complexes, which not only prolong the activity of immobilized enzymes but also are readily able to participate in electron transfer reactions. Many environmental factors can negatively affect the... 

Enzymes are biocatalysts, as such they facilitate rates of biochemical reactions. Some of the important characteristics of enzymes are summarized. Enzyme kinetics is a detailed stepwise study of enzyme catalysis as affected by enzyme concentration, substrate concentrations, and environmental factors such as temperature, pH, and so on. Two general approaches to treat initial rate enzyme kinetics, quasi-equilibrium and steady-state, are discussed. Cleland s nomenclature is presented. Computer search for enzyme data via the Internet and analysis of kinetic data with Leonora are described. 

As mentioned before, people tend intuitively to turn to the one-variable-at-a-time technique for its conceptual simplicity, and ignore the possible interaction between independent variables. A good example of the interaction between factors is that between enzyme concentration (E) and reaction temperature (T). Assuming E and T are the chosen factors for optimization, one possible interaction will be that T tends to influence the way E affects the conversion yield and vice versa. Since reaction temperature increased, enzyme activity was suppressed than at low temperature and the rate of enzyme-catalysis is affected by temperature this will inevitably affect conversion yield of the product. Should the interaction be minor or negligible, a one-factor-at-a-time search will give a satisfactory result. 

More than 2000 enzymes have been studied, each of which has a unique structure, substrate specificity, and reaction mechanism. Each reaction mechanism is affected by the catalysis-promoting factors of temperature and pH. The mechanisms of a variety of enzymes have been investigated intensively over the past several decades. The catalytic mechanisms of two well-characterized enzymes follow. 

Enzyme-based detection often relies on a change in the rate of production of a measurable product of enzymatic catalysis. Detection of an enzyme substrate is based on comparing the rate of change in product concentration in an unknown concentration of analyte to that in a known concentration of analyte. Detection of an enzyme inhibitor is accomplished using the difference in the rate of change in product concentration in the absence and presence of the inhibitor for a known substrate concentration. The rate of catalysis in enzyme based sensors can be affected by factors such as pH, temperature, or ionic... 

In the past decade or so, lipase-catalyzed esterifications and transesterifications in anhydrous media (e.g., organic solvents and supercritical fluids) have been an area of intensive research. In particular, the use of organic solvents, which normally allow a higher stability of enzymes than in water (Bock, Jimoh, Wozny, 1997), has been demonstrated. Reviews of the applications have been made by Hail Krishna and Karanth (2002) and Gandhi et al. (2000), dealing with fundamental and practical aspects of lipase catalysis. In particular, they concentrated on various immobilization strategies and factors (e.g., temperature, reaction medium, water activity) as weU as the methods of preparation (which affect and influence the stability of the lipases). 

Several key factors in enzymatic catalysis include their activity, stability, substrate concentration and temperature. Optimization of these factors will steer the enzyme towards the desired activity. Media pH was shown to affect the ionizable groups at the active site of the enzyme. Gangoiti et reported that P(3HO) depolymerase showed its maximum activity at pH 9.5 and a temperature of 40 °C. 

Proline is one amino acid which would be expected to profoundly influence the reactivity of a peptide substrate. Proline restricts the possible conformations of a peptide chain and in addition is unable to act as a hydrogen bond donor. Both of these factors could affect peptide bond cleavage by hindering substrate binding or by preventing proper catalysis. Alternately, favorable interaction could take place between the enzyme and a prolyl residue, due either to a favorable hydrophobic interaction with the prolyl side chain or because proline restricts the peptide conformation to one which is favorable. That these effects are important is accentuated by the fact that at every subsite the cleavage probability of a substrate with a proline residue is significantly different from the mean (0.148). This is true for no other amino acid residue. Proline is favorable at P4 and P3 and unfavorable at all other subsites (P2 P3) ... 

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