Abstract:
:Do non-active-site residues participate in protein function in a more direct 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, which is a mutant of aspartate aminotransferase created by directed evolution, is an ideal system because it shows a 10(6)-fold increase in the catalytic 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 function, we divided the mutations into four groups, namely, three clusters and the remainder, based on their locations in the three-dimensional structure. Mutants with various combinations of the clusters were constructed and analyzed, and the data were interpreted in the context of the structure-function relationship of this enzyme. Each cluster shows characteristic effects: for example, one cluster appears to enhance the catalytic efficiency by fixing 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 modified by the accumulation of many seemingly inert mutations of non-active-site residues.
journal_name
J Biochemjournal_title
Journal of biochemistryauthors
Shimotohno A,Oue S,Yano T,Kuramitsu S,Kagamiyama Hdoi
10.1093/oxfordjournals.jbchem.a002941keywords:
subject
Has Abstractpub_date
2001-06-01 00:00:00pages
943-8issue
6eissn
0021-924Xissn
1756-2651journal_volume
129pub_type
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