Allosteric interaction of nucleotides and tRNA ala with E. coli alanyl-tRNA synthetase

John David Dignam, Jingshu Guo, Wendell P. Griffith, Nichola C. Garbett, Amanda Holloway, Timothy Mueser

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)9886-9900
Number of pages15
JournalBiochemistry
Volume50
Issue number45
DOIs
StatePublished - Nov 15 2011

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