title: Insertion of new sequences into the catalytic domain of an enzyme.
abstract: Activities of enzymes can be modified by the replacement of active-site amino acids with residues that strengthen specific interactions with substrates or that alter the specificity. The scope for engineered enzymes would be broadened if additional, new sequences could be inserted into a catalytic domain. Properly designed, these sequences could encode new ligand binding sites, be intermediates in the construction of chimeric enzymes, or alter the internal flexibility and "breathing" modes of the active-site region. As a first step toward this objective, we inserted oligopeptides of up to 14 amino acids into various locations within an 82 amino acid region of the adenylate synthesis domain of Escherichia coli methionyl-tRNA synthetase. These sites include ones that are flanked by sequences that are conserved between the proteins from E. coli and the yeast Saccharomyces cerevisiae and those that are essential for activity and stability. We found that all of the insertional mutants are stable and some have catalytic parameters for adenylate synthesis that are comparable to those of the wild-type enzyme. Thus, such an approach may provide for a variety of novel applications.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
authors.forename: R M
authors.initials: RM
authors.lastname: Starzyk
authors.forename: J J
authors.initials: JJ
authors.lastname: Burbaum
authors.forename: P
authors.initials: P
authors.lastname: Schimmel
mesh_terms: D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid
chemical_list: D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase