Selectivity of enzymes

It is apparent that the use of enzymatic catalysis continues to grow Greater availabiUty of enzymes, development of new methodologies for thek utilization, investigation of enzymatic behavior in nonconventional environments, and the design and synthesis of new biocatalysts with altered selectivity and increased stabiUty are essential for the successhil development of this field. As more is learned about selectivity of enzymes toward unnatural substrates, the choice of an enzyme for a particular transformation will become easier to predict. It should simplify a search for an appropriate catalyst and help to estabhsh biocatalytic procedures as a usehil supplement to classical organic synthesis. 

The outstanding influence of the anionic component on the activity and selectivity of enzymes was demonstrated in the Candida rugosa lipase-catalyzed kinetic resolution of ibuprofen, a nonsteroidal antiinflammatory drug with sales of USD 183 million in 2006 (Scheme 5.15) [63]. 

Biocatalytic fuel cells using isolated redox enzymes were first investigated in 1964 [4], These fuel cells represent a more realistic opportunity for provision of implantable power, given the exquisite selectivity of enzyme catalysts, their activity under physiological conditions, and the relative ease of immobilization of isolated enzymes,... 

Electrochemical biosensors are analytical devices in which an electrochemical device serves as a transduction element. They are of particular interest because of practical advantages, such as operation simplicity, low expense of fabrication, and suitability for real-time detection. Since the first proposal of the concept of an enzyme-based biosensor by Clark, Jr [1], significant progress in this field has been achieved with the inherited sensitivity and selectivity of enzymes for analytical purposes. 

The binding sites of most enzymes and receptors are highly stereoselective in recognition and reaction with optical isomers (J, 2 ), which applies to natural substrates and synthetic drugs as well. The principle of enantiomer selectivity of enzymes and binding sites in general exists by virtue of the difference of free enthalpy in the interaction of two optical antipodes with the active site of an enzyme. As a consequence the active site by itself must be chiral because only formation of a diasteromeric association complex between substrate and active site can result in such an enthalpy difference. The building blocks of enzymes and receptors, the L-amino acid residues, therefore ultimately represent the basis of nature s enantiomer selectivity. 

Eor some recent reviews on HTS, see (a) Aharoni, A., Griffiths, A.D. and Tawfik, D.S., High-throughput screens and selections of enzyme-encoding genes. Curr. Opin. Chem. Biol., 2005, 9, 210-216 (b) Reymond, J. and Babiak, P., Screening systems. Adv. Biochem. Eng BiotechnoL, 2007,105, 31-58. 

We have chosen to explore enzymes in themK)philic anaerobic bacteria, because these kinds of microorganisms were believed to have been the first forms of life on earth and have evolved under energy limited conditions that place stress on selection of enzymes with high catalytic efficiency (2). Thermoanaerobes contain a diverse array of enzymes with unique properties and their enzyme outfits now serve as models for understanding the biodegradation of polymers such as cellulose (5). 

Carbon dioxide fixation is catalyzed by the enzyme ribulosebisphosphatecar-boxylase oxygenase (RuBisCO), which is the most abundant natural enzyme worldwide, and the least selective. In fact, RuBisCO does not follow the usual selectivity of enzymes, and at the same time promotes the carboxylation of ribulose (a C-5 sugar) to afford a C-6 sugar (with carbon fixation) and the oxidation of the same C-5 sugar, with a selectivity close to 50% [2]. 

Advantage 5 Altered Selectivity of Enzymes in Organic Solvents... 

Soon after the initial discovery, it became apparent that neither the source of the enzyme, nor the type of enzyme, nor the type of solvent seem to constrain the use of organic solvents (Zaks, 1986a). Various types of enzymes, such as lipases, proteases (chymotrypsin, subtilisin), oxidoreductases (alcohol dehydrogenase, oxidases, and peroxidases), and others, react in organic solvents. A selection of enzymes... 

Table 12.2 A selection of enzymes active in organic solvents.

Alcohol oxidase could be immobilized onto DEAE-sepharose (Pharmacia) and had very high stability even when dried and rehydrated. We therefore tried to stabilize alcohol oxidize on a solid surface and as free enzyme using DEAE dextran. However this was not successful (Fig. 5). We also knew that lactitol had been used in stabilizing various antibodies during the manufacture of antibody based tests, however lactitol also had poor stabilizing properties when dried with alcohol oxidase (Fig 5). Unexpectedly, however, we found that a combination of DEAE dextran and lactitol gave excellent stabilization of die enzyme (Fig 5.). Further experiments showed that the combination of stabilizers was generic and would be used to stabilize broad selection of enzymes (Table 1 and see ref. 17). 

Many molecules including biopolymers participate in biological functions as a molecular assembly or tissue the self-assembly of the microtublin of bacterial flagella, antigen-antibody reactions, the high activity and selectivity of enzymes, etc. are skillfully and accurately achieved by intermacromolecular interactions. 

As has already been mentioned, some lipophilic rifamycins and some strepto-varicins and geldanamycins affect the growth of cells transformed by RNA tumour viruses or the RNA-dependent DNA polymerase (reverse transcriptase) characteristic of these viruses. Again, high drug concentrations are needed to produce an effect and only partial, but never absolute, selectivity of enzyme inhibition has been found. 

This strategy has also been applied for the selection of active //-lactamases from a library of mutants also containing penicillin-binding proteins. For this purpose, the protocol had to be modified to circumvent a difficulty of selections with suicide substrates in mechanisms involving a covalent intermediate. If inhibition arises from a covalent intermediate (Y in Scheme 5.2, an acyl-enzyme in the case of serine //-lactamases), enzymes whose rate of release of this intermediate (hydrolysis of the acyl-enzyme) is slow will be efficiently selected as the efficiency of inhibition depends on the ratio of rate constants k4/k3 (Scheme 5.2). To prevent the selection of enzymes with inadequate turnover, a counter-selection step was included in the protocol the library of mutants was incubated with substrate in order to block them as covalent intermediates before adding the biotinylated inhibitor. The library could be enriched from 6 ppm to 25 % active //-lactamases in four rounds of selection [62]. 

More generally, there has been an increasing interest for reversible covalent reactions that can occur in aqueous, buffered solutions, at near physiological pH. Great progress has been made in recent years to develop biocompatible reversible chemistries. This was and remains a necessary step toward a broader use of DCC, for instance for the selection of enzyme inhibitors or nucleic acid binding ligands. 

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