Kinetic hydrolytic enzymes

The use of ILs as solvents for biotransformation reactions is, of course, not limited to kinetic resolutions and the use of hydrolytic enzymes. Numerous other... 

However, the most common and important method of synthesis of chiral non-racemic hydroxy phosphoryl compounds has been the resolution of racemic substrates via a hydrolytic enzyme-promoted acylation of the hydroxy group or hydrolysis of the 0-acyl derivatives, both carried out under kinetic resolution conditions. The first attempts date from the early 1990s and have since been followed by a number of papers describing the use of a variety of enzymes and various types of organophosphorus substrates, differing both by the substituents at phosphorus and by the kind of hydroxy (acetoxy)-containing side chain. 

Enzymes that perform the same catalytic function are known as homologous enzymes and fall into two classes. Heteroenzymes are derived from different sources and although they catalyse the same reaction they show different physical and kinetic characteristics. The hydrolytic enzyme a-amylase (EC 3.2.1.1) is found in the pancreatic secretion in man and is different from the enzymes of the same name which are derived from bacteria or malt. Iso-enzymes, sometimes referred to as isozymes, are different molecular forms of the same enzyme and are found in the same animal or organism although they often show a pattern of distribution between tissues. 

The use of enzymes and whole cells as catalysts in organic chemistry is described. Emphasis is put on the chemical reactions and the importance of providing enantiopure synthons. In particular kinetics of resolution is in focus. Among the topics covered are enzyme classification, structure and mechanism of action of enzymes. Examples are given on the use of hydrolytic enzymes such as esterases, proteases, lipases, epoxide hydrolases, acylases and amidases both in aqueous and low-water media. Reductions and oxidations are treated both using whole cells and pure enzymes. Moreover, use of enzymes in sngar chemistiy and to prodnce amino acids and peptides are discnssed. 

Figure 2.2 Production of enantiopure compounds using hydrolytic enzymes. In (a) a prochiral diester is hydrolysed to yield predominance (in theory 100%) of one enantiomer. In the next example (b) a raeso-diester is hydrolysed to yield predominance (in theory 100%) of one enantiomer of the monoester. If kj>k2 the (IS, 2i )-enantiomer is formed to the greatest extent. Due to the preference of the enzyme k4>kj and the lower monoester (IR, 2S) will be removed fastest. Hence both steps will lead to an increase of the upper enantiomer at the monoester stage. If the reaction proceeds to completion, however, the result will be another raeio-compound, a diol. In example (c) a racemic secondary ester is resolved by hydrolysis. One monoester is hydrolysed faster than the other and this leads to kinetic resolution.

The shapes of the curves in Fig. 6 are consistent with a two-step pathway, analogous to that of a hydrolytic enzyme such as a-chymotrypsin,30 in which an initial acylation burst is followed by a slow deacylation reaction. Following a fast preequilibrium binding, the first kinetic step can be attributed to acylation by substrate of the polymer imidazole residue, accompanied by simultaneous release of nit-rophenol(ate). The succeeding kinetic step would then be ascribed to hydrolysis of the acylimidazole leading to carboxylate ion and regenerated imidazole. 

Lipases are unusual hydrolytic enzymes because they act on substrates providing an interface (with few exceptions). This feature has been historically used to distinguish lipases from esterases, the latter of which act on substrates in true solution (Jensen, 1983). The distinction of lipases as interfacial catalysts can make kinetic characterization a challenge, because relevant substrate concentrations are expressed in terms of area and not concentration. 

Biocatalysts, mainly hydrolytic enzymes and oxidoreductases, have been used for organic reactions due to their excellent enantioselectivities and environmentally friendliness.1 Typical enzymatic reactions used for the organic synthesis are shown in Figure 1. Especially, hydrolytic enzymes for kinetic resolutions of racemates have been utilized widely because of their high stabilities, wide substrate specificities, lack of cofactor requirements and high availabilities. 

DYNAMIC KINETIC RESOLUTION USING HYDROLYTIC ENZYMES... 

Oxidoreductases such as dehydrogenases are also used for kinetic resolution. Selectivity of oxidoreductases is relatively high compared with that of hydrolytic enzymes. However, these enzymes have a drawback that the number of available and useful enzymes or microorganisms is still limited, and finding of new types of oxidoreductases that exhibit wide substrate specificities with high selectivities is awaiting. 

Laidler, K.J. "Some Kinetic and Mechanistic Aspects of Hydrolytic Enzyme Action" Discussion Farady Soc. 1955, 20, pp. 83-95. 

Hydrolytic enzymes that release two products in a defined sequence require a slightly more complicated model for their kinetic behavior. These enzymes pass through two intermediate stages in the catalytic cycle, E S and E S, and the formation of products involves two steps with associated rate constants k2 and k3 ... 

A second example of the use of directed molecular evolution for natural product synthesis is the use of lipases by Reetz and colleagues. This work is based on the kinetic hydrolytic resolution of racemic mixtures, in which one enantiomer is preferentially hydrolyzed and the chiral product is thus enriched. Utilizing both random mutagenesis and directed techniques such as CAST,64 they have improved the stereoselectivity of a lipase from Pseudomonas aeruginosa (PAL) on a number of occasions with different substrates. One of the first examples utilized the model substrate 2-methyldecanoic acid /xnitrophenyl ester, for which the wild-type enzyme has an enantioselectivity of E= 1.1. As a consequence of five mutations accumulated through random mutagenesis, followed by saturation mutagenesis, the enantioselectivity was increased to 25.8.123 More... 

Hydrolytic enzymes have long been used for the kinetic resolution of racemic alcohols and carboxylic acids. In recent years, the corresponding transformation on Table 14,1 List of suppliers of enzymes referred in this chapter. 

Reactants don t have to be (very) water soluble - use different solvent, or just undissolved solids Changes in solvation alter equilibria and kinetics - e. g. readily available hydrolytic enzymes catalyse synthetic reactions (including direct reversal of hydrolysis)... 

Nicotinamide-nucleotide-linked dehydrogenases were among the earliest two-substrate enzymes to be subjected to detailed kinetic study by steady-state 1-3) and rapid reaction techniques (4), and provided much of the original stimulus for the necessary extension of kinetic theory already developed for one-substrate and hydrolytic enzymes S-8). This was partly because of the convenience and precision with which rates can be measured by means of the light absorption or fluorescence emission 9-11) of the reduced coenzymes and because of the changes of these properties which accompany the binding of reduced coenzymes to many dehydrogenases 12,13). 

There are two distinct classes of hydrolytic enzymes those which have a reduced —SH group as part of their active center and others which do not have such a group. Trypsin and chymotrypsin are among the latter, while ficin and papain are among the former. We have taken up the study of ficin-catalyzed reactions side by side with our studies on trypsin because it was obvious that the two enzymes catalyze the same reaction via a different mechanism. On comparing our results for ficin (4,12) with those of Smith, Finkle, and Stockell (S) as well as with some of our own on papain, we find that from the point of view of kinetics and mechanism they appear to be very closely related enzymes. In the subsequent discussion, we assume that all that is said about ficin appUes equally to papain and probably also to other plant —SH peptidases. 

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