Enzyme hydration

It was postulated that the differences in enzyme activity observed primarily result from interactions between enzyme-bound water and solvent, rather than enzyme and solvent. As enzyme-associated water is noncovalently attached, with some molecules more tightly bound than others, enzyme hydration is a dynamic process for which there will be competition between enzyme and solvent. Solvents of greater hydrophihcity will strip more water from the enzyme, decreasing enzyme mobility and ultimately resulting in reversible enzyme deactivation. Each enzyme, having a unique sequence (and in some cases covalently or noncovalently attached cofactors and/or carbohydrates), will also have different affinities for water, so that in the case of PPL the enzyme is sufficiently hydrophilic to retain water in all but the most hydrophilic solvents. 

The amount of water in the reaction mixture can be quantified in different ways. The most common way is to nse the water concentration (in mol/1 or % by volume). However, the water concentration does not give much information on the key parameter enzyme hydration. In order to have a parameter which is better correlated with enzyme hydration, researchers have started to nse the water activity to quantify the amount of water in non-conventional reaction media (Hailing, 1984 Bell et al, 1995). For a detailed description of the term activity (thermodynamic activity), please look in a textbook in physical chemistiy. Activities are often very nselul when studying chemical equilibria and chemical reactions of all kinds, but since they are often difficult to measure they are not used as mnch as concentrations. Normally, the water activity is defined so that it is 1.0 in pure water and 0.0 in a completely dry system. Thus, dilute aqueous solutions have water activities close to 1 while non-conventional media are found in the whole range of water activities between 0 and 1. There is a good correlation between the water activity and enzyme hydration and thns enzyme activity. An advantage with the activity parameter is that the activity of a component is the same in all phases at eqnihbrium. The water activity is most conveniently measnred in the gas phase with a special sensor. The water activity in a liqnid phase can thns be measured in the gas phase above the liquid after equilibration. 

It is thus beneficial to work at fixed water activity in studies of the influence of solvents, snpports or other snbstances on enzymatic catalysis. Otherwise the effects due to differences in enzyme hydration will strongly influence the results and mask the effects songht. A typical example of this was seen when reaction rates were compared for the same reaction carried out in different solvents at varying water concentrations. In the different solvents, maximal reaction rate was observed at widely different water concentrations. However, when water was quantified in terms of water activity the optimum was observed at about the same water activity in all solvents (Valivety, Hailing and Macrae, 1992) (Figure 9.5). 

To illustrate this a model transesterification reaction catalyzed by subtilisin Carls-berg suspended in carbon dioxide, propane, and mixtures of these solvents under pressure has been studied (Decarvalho et al., 1996). To account for solvent effects due to differences in water partitioning between the enzyme and the bulk solvents. Water sorption isotherms were measured for the enzyme in each solvent. Catalytic activity as a function of enzyme hydration was measured, and bell-shaped curves with maxima at the same enzyme hydration (12%) in all the solvents were obtained. The activity maxima were different in all media, being much higher in propane than in either CO2 or the mixtures with 50 and 10% CO2. Considerations based on the solvation ability of the solvents did not offer an explanation for the differences in catalytic activity observed. The results suggest that CO2 has a direct adverse effect on the catalytic activity of subtilisin. 

The amounts of water associated with various components in a typical reaction mixture are shown in Table 1.2. Most of the water is dissolved in the reaction medium, and the amount of water bound to the enzyme is obviously just a minor fraction of the total amount of water. If the solvent was changed to one able to dissolve considerably more water and the same total amount of water was present in the system, the amount of water bound to the enzyme would decrease considerably and thereby its catalytic activity as well. Changing solvent at fixed water activity would just increase the concentration of water in the solvent and not the amount bound to the enzyme. Comparing enzyme activity at fixed enzyme hydration (fixed water activity) is thus the proper way of studying solvent effects on enzymatic reactions. 

The catalytic activity of an enzyme in an organic medium often varies by several orders of magnitude depending on the degree of enzyme hydration [9,19]. Control of enzyme hydration or water activity is thus a key issue when optimizing enzymatic conversions in organic solvents. 

These observations reveal that enzyme hydration below monolayer values has an effect on enzyme activity, and in some cases such activation can be dramatic. However, in all these examples, water was added and this may not be appropriate where nearly anhydrous reaction conditions are necessary. 

As has been stated before, oxirane derivatives are formed as intermediates during metabolic oxidations at carbon-carbon double bonds. These epoxides (arene oxides) undergo spontaneous isomerization to phenols, or enzymic hydration via epoxide hydrase to trans- dihydrodiols, or reaction with reduced glutathione (GSH) via specific GSH-transferases to the corresponding conjugates (Scheme 11), which eventually appear in urine... 

Further investigations of the reaction suggest that the bidentate complex (154) is the reactive species which undergoes external attack by water or other nucleophiles present in solution. The enzymic hydration of epoxides and the possible role of metal ions has been discussed 501 the results obtained suggest that a metal ion is not involved at the active site of epoxide hydrase. 

It has been shown that, in supercritical carbon dioxide, increases in water concentration result in increases in enzyme activity. The amount of added water needed for this increase varies and can depend on many factors, such as reaction type, enzyme utilized, and initial water content of the system. This is true until an optimal level is reached. For hydrolysis reactions, activity will either continue to increase or maintain its value. For esterification or transesterification reactions, once the optimal level of hydration has been reached, additional water will promote only side reactions such as hydrolysis. Dumont et al. (1992) suggests that additional water beyond the optimal level needed for enzyme hydration may also act as a barrier between the enzyme and the reaction medium and thereby reduce enzyme activity. Mensah et al. (1998) also observed that water above a concentration of 0.5 mmol/g enzyme led to lower catalytic activity and that the correlation between water content of the enzyme and reaction rate was independent of the substrate concentrations. 

Ebert, C., Gardossi, L., and Linda, R, Control of enzyme hydration in penicillin amidase catalyzed synthesis of amide bond, Tetrahed. Lett., 37, 9377-9380, 1996. 

Parker, M. C., Moore, B. D., and Blacker, A. J., Measuring enzyme hydration in nonpolar organic solvents using NMR, Biotechnol. Bioeng., 46, 452-458, 1995. 

Ebert C, Gardossi L, Linda P (1996) Control of enzyme hydration in peniciUin acylase catalysed synthesis of amide bond. Tetrahed Lett 37(52) 9377-9380 Elander RP (2003) Industrial production of P-lactam antibiotics. Appl Microbiol Biotechnol 61(5-6) 385-392... 

Such transformations constitute the basis of thermodynamic cycles, which have been used extensively for evaluating the free energy of binding of ligands to an enzyme, hydration free energies of small molecules, enzymatic reactions, etc. 

The strain DS5 system produced more keto product from palmitoleic and oleic acids and more hydroxy product from myristoleic, linoleic, and a- and y-linolenic acids. The reason for fliis preference is not clear. Among die 18-carbon unsaturated fatty acids, an additional double bond in either side of die C-10 position lowers the enzyme hydration activity. A hterature search revealed diat all microbial hydratases hydrate oleic and linoleic acids at the C-10 position (Fig. 2). Therefore, die positional specificity of microbial hydratases might be universal. 

Additional work has localized the oxidative activity in the particulate fraction of liver homogenate. The optimum pH is about 7, and maximal activity can be achieved in an aerobic atmosphere with the addition of TPNH. The need for oxygen and the reduced pyridine nucleotide is strong indication that hydroxylases are implicated. The probable first step in the oxidation is the formation of the primary alcohol. Beyond this point the further oxidation of the alcohol undoubtedly proceeds by routine enzymic hydration and dehydrogenations to the carboxylic acids. Single carbon fragments are then liberated as COg no aldehydic or acidic intermediates have as yet been isolated. 

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