Summary
Combinations of mutations in second-shell residues can yield positive (epistasis) (1,2). For example, phosphotriesterase H254R (8x better), when combined with D233E (2x better), has a 680-fold greater activity, whereas Kemp eliminases with beneficial active site mutations tended to see greater activity boosts from second-shell mutations than those withouts.
1.
Damry AM, Jackson CJ. The evolution and engineering of enzyme activity through tuning conformational landscapes. Protein Engineering, Design and Selection. 2021;34. Available from: https://doi.org/10.1093/protein/gzab009
2.
Zarifi N, Asthana P, Doustmohammadi H, Klaus C, Sanchez J, Hunt SE, et al. Distal mutations enhance catalysis in designed enzymes by facilitating substrate binding and product release. Nature Communications. 2025;16(1). Available from: https://doi.org/10.1038/s41467-025-63802-7