Biomaterial Database

Amino acid substitutions at position 73 in motif 2 of Escherichia coli alanyl-tRNA synthetase. 1993

S J Filley, and K A Hill

Department of Biochemistry, Loma Linda University School of Medicine, California 92350.

Lysine73, located in the adenylate synthesis domain of Escherichia coli alanyl-tRNA synthetase (AlaRS), was previously indicated to be an important residue for the interaction of this enzyme with the acceptor stem of its cognate tRNA (tRNA(Ala)). Replacement of this residue with glutamine produced a reduction in the catalytic efficiency of AlaRS in the aminoacylation assay, primarily through an increase in the apparent KM for tRNA(Ala) [Hill, K., and Schimmel, P. (1989) Biochemistry 28, 2577-2586]. Studies on the role of residue 73 in the interaction of AlaRS with its substrates have now been extended using the additional substitutions of asparagine, alanine, and glutamate. Analysis of each substituted enzyme in the ATP-PPi exchange and aminoacylation reactions reveals kinetic characteristics similar to those obtained with the glutamine substitution, except that the glutamate substitution causes a fivefold decrease in the affinity for alanine. These data verify that the positive charge on lysine 73, rather than its hydrophilic side chain, is of importance in the binding of the cognate tRNA, but do not support an ionic interaction of this residue with the RNA phosphate backbone. The collective data support the prediction that lysine73 is in motif 2 of AlaRS [Cusack, S., Hartlein, M., and Leberman, R. (1991) Nucleic Acids Res. 19, 3489-3498], but question the predicted alignment of this motif with other enzymes in its class.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008239	Lysine	An essential amino acid. It is often added to animal feed.	Enisyl,L-Lysine,Lysine Acetate,Lysine Hydrochloride,Acetate, Lysine,L Lysine
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D000412	Alanine-tRNA Ligase	An enzyme that activates alanine with its specific transfer RNA. EC 6.1.1.7.	Alanyl T RNA Synthetase,Ala-tRNA Ligase,Alanyl-tRNA Synthetase,Ala tRNA Ligase,Alanine tRNA Ligase,Alanyl tRNA Synthetase,Ligase, Ala-tRNA,Ligase, Alanine-tRNA,Synthetase, Alanyl-tRNA
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D016297	Mutagenesis, Site-Directed	Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion.	Mutagenesis, Oligonucleotide-Directed,Mutagenesis, Site-Specific,Oligonucleotide-Directed Mutagenesis,Site-Directed Mutagenesis,Site-Specific Mutagenesis,Mutageneses, Oligonucleotide-Directed,Mutageneses, Site-Directed,Mutageneses, Site-Specific,Mutagenesis, Oligonucleotide Directed,Mutagenesis, Site Directed,Mutagenesis, Site Specific,Oligonucleotide Directed Mutagenesis,Oligonucleotide-Directed Mutageneses,Site Directed Mutagenesis,Site Specific Mutagenesis,Site-Directed Mutageneses,Site-Specific Mutageneses