Hydration, enzyme activity

This enzyme, sometimes also called the Schardinger enzyme, occurs in milk. It is capable of " oxidising" acetaldehyde to acetic acid, and also the purine bases xanthine and hypoxanthine to uric acid. The former reaction is not a simple direct oxidation and is assumed to take place as follows. The enzyme activates the hydrated form of the aldehyde so that it readily parts w ith two hydrogen atoms in the presence of a suitable hydrogen acceptor such as methylene-blue the latter being reduced to the colourless leuco-compound. The oxidation of certain substrates will not take place in the absence of such a hydrogen acceptor. 

We are applying the principles of enzyme mechanism to organometallic catalysis of the reactions of nonpolar and polar molecules for our early work using heterocyclic phosphines, please see ref. 1.(1) Here we report that whereas uncatalyzed alkyne hydration by water has a half-life measured in thousands of years, we have created improved catalysts which reduce the half-life to minutes, even at neutral pH. These data correspond to enzyme-like rate accelerations of >3.4 x 109, which is 12.8 times faster than our previously reported catalyst and 1170 times faster than the best catalyst known in the literature without a heterocyclic phosphine. In some cases, practical hydration can now be conducted at room temperature. Moreover, our improved catalysts favor anti-Markovnikov hydration over traditional Markovnikov hydration in ratios of over 1000 to 1, with aldehyde yields above 99% in many cases. In addition, we find that very active hydration catalysts can be created in situ by adding heterocyclic phosphines to otherwise inactive catalysts. The scope, limitations, and development of these reactions will be described in detail. 

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. 

Why did nature use an Fe-S cluster to catalyze this reaction, when an enzyme such as fumarase can catalyze the same type of chemistry in the absence of any metals or other cofactors One speculation would be that since aconitase must catalyze both hydrations and dehydrations, and bind substrate in two orientations, Fe in the comer of a cubane cluster may provide the proper coordination geometry and electronics to do all of these reactions. Another possibility is that the cluster interconversion is utilized in vivo to regulate enzyme activity, and thus, help control cellular levels of citrate. A third, but less likely, explanation is that during evolution an ancestral Fe-S protein, whose primary function was electron transfer, gained the ability to catalyze the aconitase reaction through random mutation. 

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. 

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. 

Sometimes it can be difficult to know if the system has come to a true equilibrium concerning water distribution. It has been noted that water adsorption isotherms sometimes show hysteresis effects, which means that the water content, for example, that bound to the enzyme, depends not only on the water activity, but also on the hydration history [6]. More water is thus bound if a specified water activity is approached from a higher value (dehydration direction) than if the enzyme is hydrated from a drier state. The hysteresis effects might be due to slow conformational changes in the enzyme. 

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. 

For every turnover cycle of C02 hydration, an H+ ion must be transferred from the active site to the reaction medium. In this case, H + ion donor is the enzyme activity-linked group and the acceptor is H20 and according to Eigen and Hammes (152) the rate of H+ ion transfer to the reaction medium, H20, is the rate of C02 hydration (103-104 s-1 at pH 7). However, the observed higher rate of C02 hydration for isoenzymes CA I and II than the rate of H + ion transfer under identical experimental conditions indicate that H+ transfer must... 

Very recently the isolation of still active carbonic anhydrase from the biocrystalline layer of hens egg shells has been reported. Crude egg shell extracts exhibited no enzyme activity. however, after removal of Ca2+ and simple gel filtration of the extracts, carbonic anhydrase activity could be measured. Further purification steps led to the isolation of two isoenzymes. The possibility of inhibition of this enzyme activity by shell components cannot be excluded although recombination of shell components after separation did not inhibit that enzyme. The isolated enzymes has an apparent molecular size of 28,000 daltons. Carbonic anhydrase from egg shells catalyzes the reversible hydration of CO2 + HOH H+ + HCO3-. This probably is the primary action of the enzyme of the shell. Moreover, egg shell carbonic anhydrase catalyzes the hydration of acetaldehyde and pyridine aldehyde. Furthermore, the same enzymes have esterase activity (hydrolysis of p-nitrophenyl acetate). Whether the latter activities play a role in the egg shell cannot be judget at the present time. 

Kohlmann. K.L., et al.. Enhanced Enzyme Activities on Hydrated Lignocelluiosic Substrates, in Enzymatic Degradation of Insoluble Carbohydrates. ACS Symp. Ser. No. 618, J.N. Saddler and M.H. Penner. eds.. 1995. pp. 237-255. Kohlmann, K.L., et al. Enhanced Enzyme Activities on Hydrated Lignocelluiosic Substrates in Enzymatic Degradation of Insoluble Carbohydrates, ACS Synip. Ser. No. 618, J.N. Saddler and M H. Penner, eds., 1995b, pp. 238-255 Ladisch, M R and K. Dyck Dehydration of Ethanol New Approach Gives Positive Eneigy Balance, Science, 205(4409), 898- 900 (1979). 

Biological membranes present a barrier to the free transport of cations, as the hydrophilic, hydrated cations cannot cross the central hydrophobic region of the membrane which is formed by the hydrocarbon tails of the lipids in the bilayer. Specific mechanisms thus have to be provided for the transport of cations, which therefore allow for the introduction of controls. Such translocation processes may involve the active transport of cations against the concentration gradient with expenditure of energy via the hydrolysis of ATP. These ion pumps involve enzyme activity. Alternatively, facilitated diffusion may occur in which the cation is assisted to cross the hydrophobic barrier. Such diffusion will follow the concentration gradient until concentrations either side of... 

Hydrates have further applications in bioengineering through the research of John and coworkers (Rao et al., 1990 Nguyen, 1991 Nguyen et al., 1991, 1993 Phillips et al., 1991). These workers have used hydrates in reversed micelles (water-in-oil emulsions) to dehydrate protein solutions for recovery and for optimization of enzyme activity, at nondestructive and low-energy conditions. 

G. Bell, A. E. M. Janssen, and P. J. Halling, Water activity fails to predict critical hydration level for enzyme activity in polar organic solvents interconversion of water concentrations and activities, Enzyme Microb. Technol. 1997, 20, 47 477. 

We repeated and extended a part of Boyd s experiments using modem techniques and instrumentation facilities. We used two different potentized drugs, namely Mercuric chloride 30 and Mercuric iodide 30, and prepared them both in double distilled water and the usual medium of 90% ethanol. The purpose was to see whether the metal ion or the halide ion could play any individual role in altering the enzyme activity. The efficacy of the two media, water and aqueous ethanol, was also tested by these experiments. It is the experience of homeopathic physicians and pharmacists that potentized homeopathic drags prepared in aqueous ethanol keep their activity for a pretty long time. We wanted to test whether the pure aqueous preparation of a homeopathic potency could keep its activity as long as the hydrated... 

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. 

The addition of polyhydroxyl compounds to enzyme solutions have been shown to increase the stabilities of enzymes, (13,16,19,20). This is thought to be due to the interaction of the polyhydroxyl compound, (e.g. sucrose, polyethylene glycols, sugar alcohols, etc), with water in the system. This effectively reduces the protein - water interactions as the polyhydroxy compounds become preferentially hydrated and thus die hydrophobic interactions of the protein structure are effectively strengthened. This leads to an increased resistance to thermal denaturadon of the protein structure, and in the case of enzymes, an increase in the stability of the enzyme, shown by retention of enzymic activity at temperatures at which unmodified aqueous enzyme solutions are deactivated. 

Fig. 30. Comparison of ESR and enzyme activity changes with hydration. Effect of hydration on lysozyme dynamic properties. (Curve f) Log rate of peptide hydrogen exchange. (Curve g) , Enzyme activity (log uo) O, rotational relaxation time (log t ) of the ESR probe TEMPONE. From Rupley et al. (1983).

Stevens and Stevens (1979) measured the hydration dependence of glucose-6-phosphate dehydrogenase, hexokinase, fumarate hydratase (fumarase), and glucose-6-phosphate isomerase (phosphoglucose isom-erase) over the range 0.1-0.6 h. Serum albumin was used as a carrier protein to buffer the water content. The hydration isotherms of the enzymes and the serum albumin were assumed to be similar. For the first three enzymes activity was detected (0.05% of full solution activity) near 0.2 h. Activity was measurable for the isomerase at 0.15 h. In all cases, even at 0.3 h, the activity in the powder was less than 5% of the solution rate. Diffusion of substrates in the powder may be rate limiting. The amount of albumin in the powder affected the rate. 

The systematic measurements for chymotrypsin and lysozyme show that these enzymes differ in the hydration level of the onset of enzyme activity. The difference is sufficiendy great that experimental arufacts related to the methods of determining the extent and onset of the reaction cannot be the explanation. The other measurements cited above, although perhaps less cleanly interpretable, show threshold hydration levels ranging from below 0.1 to above 0.3 h. Apparendy, there is no single hydration level characterisdc of the onset of enzyme activity. This is not surprising, because the way in which water of the hydration shell enters into the enzyme reaction should depend on the mechanism of the reaction. 

Poole and Finney (1983b), using direct difference IR and laser Raman spectroscopy, studied the hydration of lysozyme. On the basis of these measurements, they suggested that there are conformational changes just below the hydration level for the onset of enzyme activity (i.e., 0.2-0.25 h). This conclusion conflicts with that of Careri et al. (1979b). Poole and Finney (1984) extended these measurements to lactalbumin. 

Some motional properties change significantly above hydration level for completion of principal changes in thermodynamic properties motion of bound ligand (ESR) enzyme activity water motions (NMR)... 

There appears to be no single pattern describing the hydration dependence of enzyme activity. Lysozyme activity is correlated with the unfreezing of surface motion at 0.25 A and also with the onset of surface percolation. There are changes in activity above 0.38 A, such as the changes found for rotational motion of TEMPONE. The hydration threshold for chymotrypsin activity, at 0.12 A, is substantially lower than that for lysozyme. A correlation with percolation is an attractive, but untested, possibility. 

---