Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of non-active site residues

Directed evolution was used to change the substrate specificity of aspartat e aminotransferase, A mutant enzyme with 17 amino acid substitutions was ge nerated that; shows a 2.1 x 10(6)-fold increase in the catalytic efficiency (k(cat)/K-m) for a non-native substrate, valine, The absorption spectrum o f the bound coenzyme, pyridoxal 5'-phosphate, is also changed significantly by the mutations. interestingly, only one of the 17 residues appears to be able to contact the substrate, and none of them interact with the coenzyme . The three-dimensional structure of the mutant enzyme complexed with a val ine analog, isovalerate (determined to 2.4-Angstrom resolution by x-ray cry stallography), provides insights into how the mutations affect substrate bi nding, The active site is remodeled; the subunit interface is altered, and the enzyme domain that encloses the substrate is shifted by the mutations. The present results demonstrate clearly the importance of the cumulative ef fects of residues remote from the active site and represent a new line of a pproach to the redesign of enzyme activity.