title: Mutational and kinetic analysis of a mobile loop in tyrosyl-tRNA synthetase.
abstract: The role of a mobile loop in tyrosyl-tRNA synthetase has been investigated by mutating each amino acid in the loop and kinetically analyzing the effect that each mutation has on the formation of the enzyme-bound tyrosyl adenylate intermediate. Kinetic analyses of mutations at three of the nine positions in the loop, K230, K233, and T234, have been reported elsewhere (Fersht et al., 1988; First & Fersht, 1993a,b). In this paper, the kinetic analyses of mutants in the remaining six positions, as well as a mutant in which the entire loop is deleted, are reported. With the exception of E235, which stabilizes the E.-[Tyr-ATP]++ and E.Tyr.ATP complexes by 1.0 and 1.2 kcal/mol, respectively, none of the remaining amino acids appears to be directly involved in the catalytic mechanism of the enzyme. Instead, mutation of these residues results in small alterations in the stability of E.Tyr.ATP, E.[Tyr-ATP]++ and E.Tyr.AMP.PPi complexes. The precise amino acid residues which stabilize each state vary, suggesting that the loop adopts different conformations in each of the complexes with the most highly constrained conformation being in the E.[Tyr-ATP]++ complex. Deletion of the loop reveals that the net effect of the loop in catalysis is two-fold: (1) to destabilize the E.Tyr.ATP complex preceding formation of the E.[Tyr.ATP]++ complex and (2) to stabilize the E.[Tyr-ATP]++ complex, indicating that the involvement of the loop in catalysis occurs at the expense of ATP-binding energy.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: MRC Unit for Protein Function and Design, University Chemical Laboratory, Cambridge, U.K.
authors.forename: E A
authors.initials: EA
authors.lastname: First
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014443:Tyrosine / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D014443:Tyrosine; D000255:Adenosine Triphosphate; D014448:Tyrosine-tRNA Ligase