Protein engineering reaction

Within the EC 3 class, the subclasses 1 and 3, which encompass enzymes working on ester and peptide bonds, respectively, are the most explored, in terms of known sources, protein characterization, enzyme production, protein engineering, reaction optimization, and industrial applications [13]. Despite being... 

Enzymes. Protein engineering has been used both to understand enzyme mechanism and to selectively modify enzyme function (4,5,62—67). Much as in protein stabiUty studies, the role of a particular amino acid can be assessed by replacement of a residue incapable of performing the same function. An understanding of how the enzyme catalyzes a given reaction provides the basis for manipulating the activity or specificity. 

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. 

It is difficult to attribute quantitatively by experiment the rate enhancements of the different factors contributing to catalysis. Protein engineering can get close to accurate answers when dealing with nonpolar interactions, especially in subsites. But analysis of mutation is at its weakest when altering residues that interact with charges (Chapter 15). The next development must be in improved methods of computer simulation. Controversies arise when there are no intermediates in the reaction because the kinetics can fit more than one mechanism. Again, computer simulation will provide the ultimate answers. 

What can we learn about mechanism from protein engineering that cannot be learned from classical enzymology Chapter 7 begins with the statement The mechanism of an enzymatic reaction is ultimately defined when all the intermediates, complexes, and conformational states of an enzyme are characterized and the rate constants for their interconversion are determined. The classical delineation of a mechanism would have been achieved when the general nature of intermediates on a pathway and the type of catalysis had been determined. But... 

The prerequisite for protein engineering studies is that the enzyme has been cloned and expressed. Further, unless only relatively crude information is required, it is essential that the structure has been solved at high resolution. Accurate structure-activity studies require even more stringent criteria absolute values of rate constants. The two following procedures, which were discussed earlier (Chapter 4, section E), must be available. Both depend on the accumulation of an enzyme-bound intermediate or product on the reaction pathway. 

The design of supramolecular catalysts may make use of biological materials and processes for tailoring appropriate recognition sites and achieving high rates and selectivities of reactions. Modified enzymes obtained by chemical mutation [5.70] or by protein engineering [5.71] represent biochemical approaches to artificial catalysts. 

Further advantages of biocatalysis over chemical catalysis include shorter synthesis routes and milder reaction conditions. Enzymatic reactions are not confined to in vivo systems - many enzymes are also available as isolated compounds which catalyze reactions in water and even in organic solvents [28]. Despite these advantages, the activity and stability of most wild-type enzymes do not meet the demands of industrial processes. Fortunately, modern protein engineering methods can be used to change enzyme properties and optimize desired characteristics. In Chapter 5 we will outline these optimization methods, including site-directed mutagenesis and directed evolution. 

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