Demonstration of the importance and usefulness of manipulating non-active-site residues in protein design

Do non-active-site residues participate in protein function in a more direc t way than just by holding the static framework of the protein molecule? If so, how important are they? As a model to answer these questions, ATB17, w hich is a mutant of aspartate aminotransferase created by directed evolutio n, is an ideal system because it shows a 10(6)-fold increase in the catalyt ic efficiency for valine but most of its 17 mutated residues are non-active -site residues, To analyze the roles of the mutations in the altered functi on, we divided the mutations into four groups, namely, three clusters and t he remainder, based on their locations in the three-dimensional structure. Mutants with various combinations of the clusters were constructed and anal yzed, and the data were interpreted in the context of the structure-functio n relationship of this enzyme. Each cluster shows characteristic effects: f or example, one cluster appears to enhance the catalytic efficiency by fixi ng the conformation of the enzyme to that of the substrate-bound form. The effects of the clusters are largely additive and independent of each other. The present results illustrate how a protein function is dramatically modi fied by the accumulation of many seemingly inert mutations of non-active-si te residues.