The Invariant Arginine In Motif 2 of ESCHERICHIA COLI Alanyl-tRNA Synthetase : Is Important For Catalysis But Not For Substrate Binding

<p>Structural motifs 2 and 3, located in the active site of class II aminoacyl-tRNA synthetases, each contain an invariant arginine thought to participate in interactions with ATP. For <em>Escherichia coli</em> alanyl-tRNA synthetase (AlaRS), sequence comparisons indicate that Arg69 should be aligned with the invariant arginine in motif 2 of other class II synthetases. Site-directed random mutagenesis has been employed to generate a set of proteins containing amino acid substitutions in a portion of motif 2 of AlaRS. In this set, only mutations at position 69 caused the enzyme to lose ability to complement growth of an <em>ala</em>S deletion strain, and proteins containing substitutions at position 69 alone are undetectable in a Western blot assay. A mutant protein containing the transposition of Arg69 with Gly71 does not complement growth, but does accumulate <em>in vivo</em> and has thus been purified. Michaelis and dissociation constants for the reaction of this protein with ATP are indistinguishable from those of the wild-type enzyme. However, this two-position displacement of the arginine causes a decrease in the <em>k</em>_cat for the ATP-PP_i exchange reaction by two orders of magnitude. These data suggest a role for the invariant arginine of motif 2 in stabilization of the transition stale during alanyladenylate synthesis.</p>

Name thesis:degree_name

Master of Science (MS)

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Biochemistry

Year

1994

Author dc:creator

• Lu, Ying

Contributors dc:contributor

• Kelvin A. W. Hill
• Charles W. Slattery
• E. Clifford Herrmann

• Biochemistry
• Escherichia Coli -- enzymology; Alanyl T RNA Synthetase -- chemistry; Alanyl T RNA Synthetase -- metabolism; Arginine; Point Mutation; Protein Structure, Secondary

Statement dc:rights

• This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Language dc:language

English

Repository record dc:identifier

https://scholarsrepository.llu.edu/etd/1407

OAI identifier oai:identifier

oai:scholarsrepository.llu.edu:etd-2178