Enzyme catalysis Influence

In terms of enzyme catalysis, the following factors are likely to influence the magnitude of the rate enhancement in enzymatic processes (a) proximity and orientation effects (b) electrostatic complementarity of the enzyme s active site with respect to the reactant s stabilized transition state configuration (c) enzyme-bound metal ions that serve as template, that alter pK s of catalytic groups, that facilitate nucleophilic attack, and that have... 

INFLUENCE OF OTHER STEPS ON THE MAGNITUDE OF OBSERVED ISOTOPE EFFECTS. As noted earlier, nonenzymatic reaction mechanisms do not involve those complexities imposed by substrate binding order, rates of substrate binding/release, as well as conformational changes that attend enzyme catalysis. As a result, the opportunity for detecting isotope effects is... 

The used oils in microemulsion systems are, with rare exception, non-polar and hydrophobic. The hydrophobicity of the oil has a strong influence on the resulting enzyme activity. This was first explained as being due to the interactions of the oil with the surfactants [85]. By now the studies of Laane and co-workers have shown that the solubility of the oil in the water pool has more influence on the enzyme activity independent of the choice of surfactant [4,46]. They established the so called log P -concept to describe the correlation between the hydrophobicity of the oil and the resulting enzyme activity. P is the distribution coefficient of the oil in the mixture of water and 1-octanol. In general, very hydrophilic oils (log P < 2) are not suitable for the enzyme catalysis in microemulsion because the activity and stability of the biocatalysts in these mixtures is... 

The surfactant mass fraction in a microemulsion defines the size of the interfacial area between the water and oil. The reaction rate of organic reactions in microemulsions can be dramatically enhanced by increasing the specific interfacial area [95]. Enzyme catalysis in microemulsions is usually not influenced by the size of the interfacial area because only a small fraction of the reverse micelles are hosting a bio-molecule. Most investigations published so far were made with low enzyme concentrations resulting in a low population of enzymes per reverse micelle. 

The pathway from enzyme-bound substrate to the transition state involves changes in the electronic configuration and geometry of the substrate. The enzyme itself is also not static. The ability to tightly bind the transition state requires flexibUity in the active site. Such flexibility has been experimentally demonstrated in many cases. A corollary to this is that the effectivity of enzyme catalysis can easily be influenced and regulated by conformational changes in the enzyme. An extensive consideration of the mechanisms of enzymes can be found in the works by J. Kraut (1988) and A. Fersht (1998). 

The first reported attempts of what was then called "absolute or total asymmetric synthesis" with chiral solid catalysts used nature (naturally ) both as a model and as a challenge. Hypotheses of the origin of chirality on earth and early ideas on the nature of enzymes strongly influenced this period [15]. Two directions were tried First, chiral solids such as quartz and natural fibres were used as supports for metallic catalysts and second, existing heterogeneous catalysts were modified by the addition of naturally occuring chiral molecules. Both approaches were successful and even if the optical yields were, with few exceptions, very low or not even determined quantitatively the basic feasibility of heterogeneous enantioselective catalysis was established. 

The probably most striking example so far of solvent influence on enzyme selectivity has been found by Yoshihiko Hirose and co-workers at the Amano Corp. in Nagoya, Japan, studying nifedipines, which provide a befitting conclusion of this chapter and a transition to the next on use of enzyme catalysis in the pharma industry. Nifedipines, structurally 1-substituted dihydropyridine mono- or diesters, are used in cardiovascular therapy, where they are termed calcium antagonists (Goldmann, 1991) the most prominent representative is Adalat from Bayer (Leverkusen, Germany). While the simplest achiral dihydropyridines can be syn-... 

Gay minerals and zeolites are interesting with respect to possibilities for geometric influences. Activation can be produced, as in enzyme catalysis, by constraining the reactive molecule, via surface complexation, in a configuration in which it is destabilized relative to that of the free molecule, yet still accessible to other reactants. A possible example is hydrazine complexed with kaolinite. The conformation of hydrazine is flattened relative to that of the free molecule (See Giff Johnston s paper in Part m of this volume). It has been shown that hydrazine is readily air-oxidized by kaolinite (Coyne, submitted for publication). 

Acid-catalyzed S—O fission may occur under neutral conditions under the influence of enzymic catalysis. Perhaps a functional carboxyl group obtains at the active site of the enzyme, a group which occurs in undissociated acid form and works as a powerful acid catalyst because of its location at a special hydrophobic pocket. However, we have examined other possibilities of metal-ion-catalyzed S—O fission. 

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. 

Substrate selectivity and stereoselectivity of enzyme catalysis are known to be influenced by the reaction media. As has been mentioned in the preceding section, a profound feature of the behaviour of enzymes in an anhydrous organic medium is the conformational stability, which leads to enhanced thermal stability and the ligand memory property. These features of enzymes have been exploited to impart novel catalytic characteristics that are absent in the native biopolymers. 

So far, only very little attention has been focussed on the use of zeolites in biocatalysis, i.e., as supports for the immobilization of enzymes. Lie and Molin [116] studied the influence of hydrophobicity (dealuminated mordenite) and hydrophilicity (zeolite NaY) of the support on the adsorption of lipase from Candida cylindracea. The adsorption was achieved by precipitation of the enzyme with acetone. Hydrolysis of triacylglycerols and esterification of fatty acids with glycerol were the reactions studied. It was observed that the nature of the zeolite support has a significant influence on enzyme catalysis. Hydrolysis was blocked on the hydrophobic mordenite, but the esterification reaction was mediated. This reaction was, on the other hand, almost completely suppressed on the hydrophilic faujasite. The adsorption of enzymes on supports was also intensively examined with alkaline phosphatase on bentolite-L clay. The pH of the solution turned out to be very important both for the immobilization and for the activity of the enzyme [117]. Acid phosphatase from potato was immobilized onto zeolite NaX [118]. Also in this study, adsorption conditions were important in causing even multilayer formation of the enzyme on the zeolite. The influence of the cations in the zeolite support was scrutinized as well, and zeolite NaX turned out to be a better adsorbent than LiX orKX. 

Influence of H +, which exists in solution as HiO+, on enzyme catalysis can be very complex and traced to the stability of the enzyme, changes in its conformation, protonation of sensitive groups (amino groups, histidine), association state of free enzyme, effects on enzyme-substrate interactions, chemical changes in ES, etc. 

As for any catalytic process, the study of enzyme catalysis focuses on the determination of reaction energies and kinetic parameters, which requires understanding ground state and transition state (TS) geometries. Computational methods are valuable in this matter, as the TS is hard to be trapped experimentally due to the short-lived nature of the species. However, the intrinsic complexity of the bio-enzymatic reaction, previously described, as well as the way it influences the kinetics have to be modeled in the calculations. 

Early reports on the isolation of a pyrophosphate lyase and a phosphatase activity from E. coli had no apparent follow up." Later, it was proposed that the release of pyrophosphate could proceed without enzyme catalysis under the influence of magnesium or calcium ions." " ... 

As already mentioned, control of vrater content is of great importance in enzyme catalysis. Studies on Pseudomonas sp. lipase have also revealed a strong influence of the vrater content of the reaction medium [70]. In order to compare the enzyme activity and selectivity as a fonction of the vrater present in solvents of different polarities, it is necessary to use the vrater activity a in these solvents. We used the method of vrater activity equilibration over saturated salt solutions [71] and could demonstrate that, in contrast to MTBE, which is commonly used for this type of reaction, the enantiosdectivity of the lipase is less influenced either by the water content or the temperature when the reaction is performed in [BMIM][(CFjS02)2N]. 

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