Document Detail

Mutational and kinetic analysis of a mobile loop in tyrosyl-tRNA synthetase.
MedLine Citation:
PMID:  8257699     Owner:  NLM     Status:  MEDLINE    
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)
E A First; A R Fersht
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  32     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  1993 Dec 
Date Detail:
Created Date:  1994-01-19     Completed Date:  1994-01-19     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  13658-63     Citation Subset:  IM    
MRC Unit for Protein Function and Design, University Chemical Laboratory, Cambridge, U.K.
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MeSH Terms
Adenosine Triphosphate / metabolism
Base Sequence
Binding Sites
Enzyme Stability
Escherichia coli / genetics
Gene Deletion
Geobacillus stearothermophilus / enzymology,  genetics
Molecular Sequence Data
Mutagenesis, Site-Directed*
Protein Structure, Secondary*
Structure-Activity Relationship
Tyrosine / metabolism
Tyrosine-tRNA Ligase / chemistry*,  genetics*,  metabolism
Grant Support
1 F32 GM12023-01/GM/NIGMS NIH HHS
Reg. No./Substance:
55520-40-6/Tyrosine; 56-65-5/Adenosine Triphosphate; EC Ligase

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