Chapter 6: Modulating Enzyme Activity via Incorporation of Non-canonical Amino Acids

In this chapter, we discuss recent applications of non-canonical amino acids in modulating enzyme activity. The methodology available for both non-specific and site-specific non-canonical amino acid incorporation into enzymes has advanced in recent years, which has allowed the novel properties of non-canonical amino acids to supplement the already impressive catalytic properties of enzymes. Here, we provide a brief introduction into the methodology underlying non-canonical amino acid incorporation, before discussing recent applications of non-canonical amino acids in developing enzymes with increased soluble expression and thermostability, enhanced catalytic efficiency, altered substrate specificity and selectivity, and to improve chemical modification of enzymes. In addition to these applied aims, we also cover the use of non-canonical amino acids to probe enzyme function and mechanism. We end this chapter by highlighting the smaller number of studies that have used non-canonical amino acid mutagenesis with enzymes, in comparison to its more frequent use in studying binding proteins or peptides, highlighting a number of technical hurdles that must be overcome before this field reaches its potential.