Effects of Organic Solvents on Enzyme Selectivity

Organic Synthesis with Enzymes in Non-Ajueous Media. Edited by Giacomo Carrea and Sergio Riva Copyright 2008 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim ISBN 978-3-527-31846-9 

Selectivity is an intrinsic properly of enzymatic catalysis. [3] Following the nomenclature proposed by Cleland [24, 25], the pseudo second-order rate constant for the reaction of a substrate with an enzyme, kml/KM, is known as the specificity constant, ksp. [26] To express the relative rates of competing enzymatic reactions, involving any type of substrates, the ratio of the specificity constants appears to be the parameter of choice [3]. Since the authoritative proposition by Sih and coworkers [27], the ratio of specificity constants for the catalytic conversion of enantiomeric substrates, R and S, is commonly known as the enantiomeric ratio or E -value (Equation 1)  

For enzymes that obey the rate equation r= ksp-cs-cE, with cs the substrate concentration and cE the concentration of free, uncomplexed, enzyme, this leads directly to Equation 2 for a catalytic reaction involving both enantiomers  

The notation ER or ERS to express the preferential conversion of R over S, has not gained widespread popularity. Any ambiguity arising from the use of the unsuperscripted notation is addressed in the accompanying text. With 0 E °° and ERS = 1 /Esr, it is common practice to express the enantiomeric ratio as / 1, with separate indication of the preferred enantiomer. 

The enantiomeric ratio is an intrinsic feature of enzyme-enantiomer couples. The actual realization of this property in a resolution reaction affects the enantiomeric excess value, ees (for the substrate) and eeP (for the product) (Equation 3)  

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Jongejan JA (2008) Effects of organic solvents on enzyme selectivity. In Carrea G, Riva S (eds) Organic Synthesis with Enzymes in Non-aqueous Media. Wiley-VCH, Weinheim, p 25... 

Nevertheless, 1 and 10 xM are often arbitrarily selected as the initial concentrations for metabolic stability assays (MacKenzie et al., 2002 Obach and Reed-Hagen, 2002). The effects of organic solvents on enzyme activities have been carefully investigated (Busby et al., 1999 Chauret et al., 1998 Easterbrook et al., 2001). For the purpose of data comparability for enzyme activities, the levels of organic vehicles should, in principle, be kept as low as possible, and consistent in all incubations in a given experiment. Dimethyl sulfoxide (DMSO) and methanol (or acetonitrile), the most commonly used vehicle solvents, should be kept at levels equal to, or preferably less than, 0.2 and 2% (v/v), respectively (Easterbrook et al., 2001 Hickman et al., 1998). 

In the following text, examples of solvent effects on enzyme selectivity, referred either to systems based (i) on water-miscible organic cosolvents added to aqueous buffers or (ii) on organic media with low water activity, are discussed. 

The choice of organic solvent can also have a dramatic effect on selectivity.In contrast to enzyme activity, in the majority of examples reported there appears to be no correlation between solvent physical properties and enantioselectivity. In fact, investigating the effect of various solvents towards a number of lipases, Secundo et al also found that the optimal solvent differed with both enzyme and substrate. A number of theories have been postulated in order to explain these effects in individual cases, but none has any general predictive value. This is somewhat intriguing given that differences in enantioselectivity simply relate to a change in the relative rate of conversion of each enantiomer. 

To assess the selectivity of our method, the fish samples have been spiked with 2 ppm of mercury (II) and then treated as described before. After toluene incubation with invertase enzyme (0.05 pg/mL) no effect has been observed on enzyme activity. The same amount of mercury (II) has been added in fish samples in presence of 0.4 ppm of methyl mercury. In this case, 50% of inhibition has been noted and corresponds exactly to the value obtained when we studied the calibration cure in absence of mercury (II) (Fig. 20.4). This result is in agreement with what is reported in the literature showing that the methyl mercury is much more soluble in organic solvents than the mercury ions [8]. In addition to this high selectivity of our method, we have shown previously that the enzyme invertase is selective for mercury [3]. In the presence of Zn2+, Cu2+, Cd2+, Pb2+ and Fe3+ no inhibition was detected for these cations. 

Haas et al. (162) have studied enzymatic phosphatidylcholine hydrolysis in organic solvents by examining selected commercially available lipases. Enzymatic hydrolysis of oat and soy lecithins, and its effect on the functional properties of lecithin, was investigated by Aura et al. (163). The phospholipase used was most effective at low enzyme and substrate concentrations. 

A more general use of proteolytic enzymes in peptide synthesis became feasible with the discovery (Sealock and Laskowsky 1969) of the effect of water miscible organic solvents on the equilibrium in enzyme catalyzed peptide bond hydrolysis and synthesis. In the presence of isopropanol (or dimethylfor-mamide, etc.) the dissociation of the carboxyl group is suppressed and, at least in a selected pH region, the equilibrium is shifted toward synthesis. A notable case is the conversion of porcine insulin to human insulin. Enzymatic cleavage of the C-terminal residue of the B-chain (alanine) with carboxypeptidase yields desalanino pork insulin. This cleavage is followed by the incorporation of... 

The specificity of enzyme reactions can be altered by varying the solvent system. For example, the addition of water-miscible organic co-solvents may improve the selectivity of hydrolase enzymes. Medium engineering is also important for synthetic reactions performed in pure organic solvents. In such cases, the selectivity of the reaction may depend on the organic solvent used. In non-aqueous solvents, hydrolytic enzymes catalyse the reverse reaction, ie the synthesis of esters and amides. The problem here is the low activity (catalytic power) of many hydrolases in organic solvents, and the unpredictable effects of the amount of water and type of solvent on the rate and selectivity. 

Lewis-acid catalysis of Diels-Alder reactions involving bidentate dienophiles in water is possible also if the beneficial effect of water on the catalyzed reaction is reduced relative to pure water. There are no additional effects on endo-exo selectivity. As expected, catalysis by Cu ions is much more efficient than specific-acid catalysis.Using a-amino acids as chiral ligands, Lewis-acid enan-tioselectivity is enhanced in water compared to organic solvents. Micelles, in the absence of Lewis acids, are poor catalysts, but combining Lewis-acid catalysis and micellar catalysis leads to a rate accelaration that is enzyme-like. 

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