Probing the Mechanism of Enzyme Action Through Specific Amino Acid Substitutions

Abstract

The technique of site-directed mutagenesis mediated by specific oligonucleotides is a rapid, simple and powerful tool with which to study the roles played by particular amino acid side chains in proteins. The information yielded from such studies is in proportion to how much is already known structurally and functionally about the molecule under investigation. For DNA gyrase of Escherichia coli the essentiality of the “active site” tyrosine 122 has been tested by making phenylalanine and serine replacements. For the tyrosyl-tRNA synthetase of Bacillus stearothermophilus, an enzyme whose three dimensional structure is known at high resolution, the subtle contributions to binding and catalysis made by residues well removed from the actual reaction center have been determined. In addition, the importance for specificity of different types of hydrogen bonds has been investigated. Studies of this enzyme have shown it is possible to engineer a mutant with enhanced substrate affinity and these and other studies indicate that there are good prospects for designing enzymes with greater stabilities and with altered substrate specificities.