Enzyme selection

Selectivity The analysis of closely related compounds, as we have seen in earlier chapters, is often complicated by their tendency to interfere with one another. To overcome this problem, the analyte and interferent must first be separated. An advantage of chemical kinetic methods is that conditions can often be adjusted so that the analyte and interferent have different reaction rates. If the difference in rates is large enough, one species may react completely before the other species has a chance to react. For example, many enzymes selectively cat-... 

A number of examples of monoacylated diols produced by enzymatic hydrolysis of prochiral carboxylates are presented in Table 3. PLE-catalyzed conversions of acycHc diesters strongly depend on the stmcture of the substituent and are usually poor for alkyl derivatives. Lipases are much less sensitive to the stmcture of the side chain the yields and selectivity of the hydrolysis of both alkyl (26) and aryl (24) derivatives are similar. The enzyme selectivity depends not only on the stmcture of the alcohol, but also on the nature of the acyl moiety (48). 

Restrictions for the substrates of the transketolase-catalyzed reaction only arise from the stereochemical requirements of the enzyme. The acceptor aldehyde must be formaldehyde9,20, glycolaldehydel6,17 or a (R)-2-hydroxyaldehyde10,17. The donor ketose must exhibit a (3(7,4 R) configuration10. The enzyme selectively adds the hydroxyacetyl moiety to the Re-face of the acceptor aldehyde leading to a 3(7 configuration of the products. 

The term medium engineering , that is the possibility to affect enzyme selectivity simply by changing the solvent in which the reaction is carried out, was coined by Klibanov, who indicated it as an alternative or an integration to protein engineering [5aj. Indeed, several authors have confirmed that the enantio-, prochiral-, and even regioselectivity of enzymes can be influenced, sometimes very remarkably, by the nature of the organic solvent used. 

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 interest and success of the enzyme-catalyzed reactions in this kind of media is due to several advantages such as (i) solubilization of hydrophobic substrates (ii) ease of recovery of some products (iii) catalysis of reactions that are unfavorable in water (e.g. reversal of hydrolysis reactions in favor of synthesis) (iv) ease of recovery of insoluble biocatalysts (v) increased biocatalyst thermostability (vi) suppression of water-induced side reactions. Furthermore, as already said, enzyme selectivity can be markedly influenced, and even reversed, by the solvent. 

The simplest way to prepare a biocatalyst for use in organic solvents and, at the same time, to adjust key parameters, such as pH, is its lyophilization or precipitation from aqueous solutions. These preparations, however, can undergo substrate diffusion limitations or prevent enzyme-substrate interaction because of protein-protein stacking. Enzyme lyophilization in the presence of lyoprotectants (polyethylene glycol, various sugars), ligands, and salts have often yielded preparations that are markedly more active than those obtained in the absence of additives [19]. Besides that, the addition of these ligands can also affect enzyme selectivity as follows. 

In all the reported examples, the enzyme selectivity was affected by the solvent used, but the stereochemical preference remained the same. However, in some specific cases it was found that it was also possible to invert the hydrolases enantioselectivity. The first report was again from iQibanov s group, which described the transesterification of the model compound (13) with n-propanol. As shown in Table 1.6, the enantiopreference of an Aspergillus oryzae protease shifted from the (l)- to the (D)-enantiomer by moving from acetonitrile to CCI4 [30]. Similar observations on the inversion of enantioselectivity by switching from one solvent to another were later reported by other authors [31]. 

The experimental evidences that medium engineering might represent an efficient method to modify or improve enzyme selectivity (alternative to protein engineering and to the time-consuming search for new catalysts) were immediately matched by the search for a sound rationale of this phenomenon. The different hypotheses formulated to try to rationalize the effects of the solvent on enzymatic enantioselectivity can be grouped into three different classes. The first hypothesis suggests that... 

However, whatever the mechanism of action is, the effect of solvents on enzyme selectivity is sometimes really dramatic. For example, Hrrose et al. [42] reported that in the Pseudomonas species lipase-catalyzed desymmetrization of prochiral... 

As shown in this chapter, by focusing on the modulation of enzyme selectivity by medium engineering, quite simple modifications of the solvent composition can really have significant effects on the performances of the biocatalysts. The main drawback remains the lack of reliable predictive models. Despite the significant research efforts (particularly in the last decade), it is likely that a reasonable foresight of the enantioselective outcome of an enzymatic transformation will continue to be based solely on a careful analysis of the increasingly numerous literature reports. 

KROGDAHL A, HOLM H (1981) Soybean proteinase inhibitors and human proteolytic enzymes selective inactivation of inhibitors by treatment with human gastric juice. /M/fr. Ill 2045-51. 

This enzyme catalyzes the conversion of pyruvate to formate and acetyl CoA and is a key enzyme in the anaerobic degradation of carbohydrates in some Enterobacteriaceae. Using an enzyme selectively C-labeled with glycine, it was shown by EPR that the reaction involves production of a free radical at C-2 of glycine (Wagner et al. 1992). This was confirmed by destruction of the radical with O2, and determination of part of the structure of the small protein that contained an oxalyl residue originating from gly-734. 

Arthrobacter sp. Ru61a Gram (+) [327,350] Susanne Fetzner Quinaldine 4-OR BDN of 2-MeQN through the anthranilic acid pathway. Activity is about 70 times improved by purification from the crude extract. Enzyme activity for QN is three times higher than for quinaldine (2Me-QN). Enzyme selective to most of heterocyclic N-compounds. Attack at the 4th (para)-position. 

The are several clearance and toxicological aspects that have to be considered in the drug discovery process such as metabolic stability, enzyme selectivity, CYP inhibition and type of inhibition. Among these factors, the prediction of the site of metabolism has become one of the most successful parameters for prediction. The knowledge of the site of metabolism enhances the opportunity to chemically modify the molecule to improve the metabolic stability. There are several approaches based on database mining, chemical reactivity, protein interaction or both that have been developed for the prediction of this property, with different degree of success and applicability. 

The following will always cause a reduction in the activity of an enzyme. Select true or false for each of the statements ... 

Enzyme hydrolysis of immobihzed substrates was not only used for the development of linkers in sohd-phase chemistry but also used as a key step to evaluate enzyme-selectivity in several assays. 

A novel continuous-flow SCCO2 process for the kinetic resolution of 1-phenyethanol enantiomers (Figure 30) using Novozym 435 immobilized enzyme from Candida antarctica was described by Matsuda et al. [51], The lipase enzyme, selectively acetylated the R)-alcohol component. A mixture of starting material and vinyl acetate was passed through the enzyme with supercritical carbon-dioxide (Figure 31). The reaction zone was pressurized and heated, so the reaction could be performed imder supercritical conditions, synthesizing the desired (i )-acetate with 99.7% ee. and 47% yield. 

The biochemical hallmark of apopfosis is fhe fragmentation of the genomic DNA, an irreversible event that commits the cells to die. This fragmentation in many cell types has been shown to result from the activation of an endogenous Ca +- and Mg +-dependent nuclear endonuclease (W18). This enzyme selectively cleaves DNA at sites located between nucleosomal units (linker DNA), generating mono- and oligonucleosomal DNA fragments. 

Drug Reversible inhibition Enzyme selectivity Indication... 

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