However, in the enzyme engineering [18] what we are more interested is that how the amino acid mutations at certain positions modify (or improve) the biological functions (including bioactivity, thermostability, pH tolerance, and other properties) of enzymes.
It has been widely recognized that the mutations at specific directions are caused by the functional constraints in protein family and the directional mutations at certain positions control the evolutionary direction of the protein family.
Counting mutations would be somewhat too simplistic because some mutations at certain positions may be very unstable (because of a site-specific mutation rate that is well above average) and therefore provide little evidence in favor of an artificial cause rather than natural evolution.
Mutations at certain residues of PTEN, that are associated with Cowden's disease, affect the ubiquitination of PTEN and prevent nuclear translocation.
Instead, the NNH>NSH "more hotspots" criterion gets at a somewhat different issue, namely the clustering of observed mutations at certain sites ("hotspots").
Partitioning the sites into groups according to ancestral amino acid identity explains some of the variation, with mutations at certain amino acids being unconditionally deleterious, others unconditionally nearly neutral, and others with substantially variable effects.
