title: Evidence from cassette mutagenesis for a structure-function motif in a protein of unknown structure.
abstract: The three-dimensional structure of most enzymes is unknown; however, many enzymes may have structural motifs similar to those in the known structures of functionally related enzymes. Evidence is presented that an enzyme of unknown structure [Ile-transfer RNA (tRNA) synthetase] may share a functionally important structural motif with an enzyme of related function (Tyr-tRNA synthetase). This approach involves (i) identifying segments of Ile-tRNA synthetase that have been unusually conserved during evolution, (ii) predicting the function of one such segment by assuming a structural relation between Ile-tRNA synthetase and Tyr-tRNA synthetase, and (iii) testing the predicted function by mutagenesis and subsequent biochemical analysis. Random mutations were introduced by cassette mutagenesis into a ten-amino-acid segment of Ile-tRNA synthetase that was predicted to be involved in the formation of the binding site for isoleucine. Few amino acid substitutions appear to be tolerated in this region. However, one substitution (independently isolated twice) increased the Michaelis constant Km for isoleucine in the adenylate synthesis reaction by greater than 6000-fold, but had little effect on the Km for adenosine triphosphate, the apparent Km for tRNA, or the rate constant kcat.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
authors.forename: N D
authors.initials: ND
authors.lastname: Clarke
authors.forename: D C
authors.initials: DC
authors.lastname: Lien
authors.forename: P
authors.initials: P
authors.lastname: Schimmel
mesh_terms: D000604:Amino Acyl-tRNA Synthetases; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology; D007533:Isoleucine-tRNA Ligase; D007700:Kinetics; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship
chemical_list: D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase