Enzyme-selective substrates

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 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. 

AP isoenzymes can cleave associated phosphomonoester groups from a wide variety of substrates. The exact biological function of these enzymes is not well understood. They can behave in vivo in their classic phosphohydrolase role at alkaline pH, but at neutral pH AP isoenzymes can act as phosphotransferases. In this sense, suitable phosphate acceptor molecules can be utilized in solution to increase the reaction rates of AP on selected substrates. Typical phosphate acceptor additives include diethanolamine, Tris, and 2-amino-2-methyl-lpropanol. The presence of these additives in substrate buffers can dramatically increase the sensitivity of AP ELISA determinations, even when the substrate reaction is done in alkaline conditions. 

Fig. 6.13. Schematic representation of a selective delivery obtained by antibody-directed en-zyme-prodrug therapy (ADEPT). An exogenous enzyme is coupled to a monoclonal antibody (mAb) targeted for tumor cells. In a second step, a prodrug is administered, which, as a selective substrate of the exogenous enzyme, will be selectively activated at the tumor site.

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. 

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. 

Fig. 8.14 Scheme of a pull-down assay. The enzymatic reaction is completely carried out in solution. Upon enzyme addition, substrate is consumed, and product is formed. Sample aliquots are taken at several time points from the reaction mixture and are taken to a SAM, which has been modified with selective end groups. The latter are able to bind both substrate and product. Finally, matrix is added, and the SAM is analyzed by means of MALDI-MS. 

Molecular complexation is a precondition for receptor functions such as substrate selection, substrate transportation, isomeric differentiation, and stereoselective catalysis. Although the investigation of such functions with synthetically derived compounds is a relatively new development in chemistry, they are well known and extensively studied functions in the biological domain. Evolution, gene expression, cell division, DNA replication, protein synthesis, immunological response, hormonal control, ion transportation, and enzymic catalysis are only some of the many examples where molecular complexation is a prerequisite for observing a biological process. 

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