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.