Role of distal sites in enzyme engineering

The limitations associated with natural enzyme catalysis have triggered the rise of the field of protein engineering. Traditional rational design was based on the analysis of protein structural information and catalytic mechanisms to identify key active sites or ligand binding sites to reshape the substrate pocket. The role and significance of functional sites in the active center have been studied extensively. With a deeper understanding of the structure-catalysis relationship map, the entire protein molecule can be filled with residues that play a substantial role in its structure and function. However, the catalytic mechanism underlying distal mutations remains unclear. The aim of this review was to highlight the criticality of the distal site in enzyme engineering based on the following three aspects: What can distal mutations exert on function from mutability landscape? How do distal sites influence enzyme function? How to predict and design distal mutations? This review provides insights into the catalytic mechanism of enzymes from the global interaction network, knowledge from sequence-structure-dynamics-function relationships, and strategies for distal mutation-based protein engineering.