Amino acid substitution enzyme techniques

Just as nature uses evolution to naturally select enzyme variations that provide an advantage to the host, directed evolution of the amino acid substitution utilize techniques to screen or select for mutant enzymes that perform better than wild-type enzymes. Unlike site-directed mutagenesis, directed evolution techniques do not require a detailed understanding of the enzyme in order to identify useful variants. The techniques of applied molecular evolution however, do require a screening or selection step to identify the individual mutants of interest (Fig. 12.2). 

Site-directed mutagenesis has become an important and widespread technique for the elucidation of structure-function relationships in proteins. However, the repercussions of mutations on both protein structure and catalysis are often subtle and, particularly in the case of mechanisms that require multiple catalytic steps, not always easily interpretable. Classical comparison of catalytic rate parameters between mutant and native enzymes where an amino acid substitution results in a change in the the rate-limiting step of a reaction are not necessarily valid (109). Thus, direct detection of reaction intermediates is an important means for assessing the effect of mutations on the mechanism and for accurately determining the role of various protein residues in catalysis. 

A comprehensive description of mutagenesis techniques and successful examples in the past decades would be a daunting work. Numerous new and enlightening techniques are piling up for accurate, fast and simple discovery of more efficient, versatile or robust enzymes. We would like to share some recent examples from our laboratory in the following sections to exhibit the prowess of amino acid substitutions for modulating enzyme activity, selectivity, and thermostability. 

However, recently it has proved possible to positively identify tryptophan radicals in cytochromec peroxidase[147] and tyrosine radicals in ribonucleotide reductase, prostaglandin H synthase and photosystem II of chloroplasts [148], This has been achieved by a combination of the techniques discussed already, but with the powerful, additional non-invasive tool of isotopic substitution. As deuterons (5=1) give different splitting than protons (S = 1/2), substituting different labelled amino-acid residues into the enzyme should reveal the nature of the radical-containing residue. This is easily achieved in an auxotrophic mutant that requires this amino acid to be supplied in the medium. The specific residue can then be identified by site-directed mutagenesis of the evolutionary conserved amino-acid residues [108,149-151]. 

Site-directed mutagenesis is an indispensable technique for determining the effect of substituting a specific amino acid writh another. For example, enzyme reaction rates can be measured for both wdld type and mutant enzymes, and changes in enzyme kinetics can be monitored to assess the possible catalytic role of a given residue. The complete absence of activity in a mutant enzyme indicates that the mutated residue is essential for catalysis. 

With the development of site-specific mutagenesis by Michael Smith in the 1970s [16], a given amino acid at a defined position in a protein can be substituted by any one of the other 19 canonical amino acids. Today mutagenesis is a commonly used technique for the study of enzyme mechanisms [17], and also indispensable in protein engineering when attempting to improve the catalytic profile of... 

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