The Invariant Arginine In Motif 2 of ESCHERICHIA COLI Alanyl-tRNA Synthetase : Is Important For Catalysis But Not For Substrate Binding
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 Escherichia coli 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 alaS 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 in vivo 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 kcat for the ATP-PPi 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.
Kelvin A. W. Hill
Charles W. Slattery
E. Clifford Herrmann
Master of Science (MS)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
Escherichia Coli -- enzymology; Alanyl T RNA Synthetase -- chemistry; Alanyl T RNA Synthetase -- metabolism; Arginine; Point Mutation; Protein Structure, Secondary
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Lu, Ying, "The Invariant Arginine In Motif 2 of ESCHERICHIA COLI Alanyl-tRNA Synthetase : Is Important For Catalysis But Not For Substrate Binding" (1994). Loma Linda University Electronic Theses, Dissertations & Projects. 1407.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives