Alanyl-tRNA synthetase, a dimeric class 2 aminoacyl-tRNA synthetase, activates glycine and serine at significant rates. An editing activity hydrolyzes Gly-tRNA(ala) and Ser-tRNA(ala) to ensure fidelity of aminoacylation. Analytical ultracentrifugation demonstrates that the enzyme is predominately a dimer in solution. ATP binding to full length enzyme (ARS875) and to an N-terminal construct (ARS461) is endothermic (ΔH = 3-4 kcal mol(-1)) with stoichiometries of 1:1 for ARS461 and 2:1 for full-length dimer. Binding of aminoacyl-adenylate analogues, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine (ASAd) and 5'-O-[N-(L-glycinyl)sulfamoyl]adenosine (GSAd), are exothermic; ASAd exhibits a large negative heat capacity change (ΔC(p) = 0.48 kcal mol(-1) K(-1)). Modification of alanyl-tRNA synthetase with periodate-oxidized tRNA(ala) (otRNA(ala)) generates multiple, covalent, enzyme-tRNA(ala) products. The distribution of these products is altered by ATP, ATP and alanine, and aminoacyl-adenylate analogues (ASAd and GSAd). Alanyl-tRNA synthetase was modified with otRNA(ala), and tRNA-peptides from tryptic digests were purified by ion exchange chromatography. Six peptides linked through a cyclic dehydromoropholino structure at the 3'-end of tRNA(ala) were sequenced by mass spectrometry. One site lies in the N-terminal adenylate synthesis domain (residue 74), two lie in the opening to the editing site (residues 526 and 585), and three (residues 637, 639, and 648) lie on the back side of the editing domain. At least one additional modification site was inferred from analysis of modification of ARS461. The location of the sites modified by otRNA(ala) suggests that there are multiple modes of interaction of tRNA(ala) with the enzyme, whose distribution is influenced by occupation of the ATP binding site.