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 Arg-69 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 Arg-69 with Gly-71 does not complement growth but does accumulate in vivo and has thus been purified. Michaelis and dissociation constants for the interaction 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 2 orders of magnitude. These data suggest a role for the invariant arginine of motif 2 in stabilization of the transition state during alanyladenylate synthesis.
Lu
Y
Y
Department of Biochemistry, Loma Linda University School of Medicine, California 92350.
Hill
K A
KA
D000412:Alanine-tRNA Ligase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001120:Arginine; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D001120:Arginine; D000412:Alanine-tRNA Ligase