Mutational analysis of a leucine heptad repeat motif in a class I aminoacyl-tRNA synthetase.
Aminoacyl-tRNA synthetases activate amino acids with ATP to form aminoacyl adenylates as the essential intermediates for aminoacylation of their cognate tRNAs. The class I Escherichia coli cysteine tRNA synthetase contains an N-terminal nucleotide binding fold that provides the catalytic site of adenylate synthesis. The C-terminal domain of the cysteine enzyme is predominantly alpha-helical and contains a leucine heptad repeat motif. We show here that specific substitutions of leucines in the leucine heptad repeats reduced tRNA aminoacylation. In particular, substitution of Leu316 with phenylalanine reduced the catalytic efficiency of aminoacylation by 1000-fold. This deleterious effect was partially alleviated by a more conservative substitution of leucine with valine. Filter binding assays show that neither the phenylalanine nor the valine substitution at Leu316 had a major effect on the ability of the cysteine enzyme to bind tRNA(Cys). In contrast, pyrophosphate exchange assays show that both substitutions decreased the adenylate synthesis activity of the enzyme. Analysis of these results suggests that the primary defect of the valine substitution is executed at adenylate synthesis while that of the phenylalanine substitution is at both adenylate synthesis and the transition state of tRNA aminoacylation. Thus, although Leu316 is located in the C-terminal domain of the cysteine enzyme, it may modulate the capacity of the N-terminal domain for amino acid activation and tRNA aminoacylation through a domain-domain interaction.
Ohannesian
D W
DW
Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Oh
J
J
Hou
Y M
YM
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D000604:Amino Acyl-tRNA Synthetases; C019975:cysteinyl-tRNA synthetase; D007930:Leucine