TY - JOUR
T1 - Allosteric interaction of nucleotides and tRNA ala with E. coli alanyl-tRNA synthetase
AU - Dignam, John David
AU - Guo, Jingshu
AU - Griffith, Wendell P.
AU - Garbett, Nichola C.
AU - Holloway, Amanda
AU - Mueser, Timothy
PY - 2011/11/15
Y1 - 2011/11/15
N2 - 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.
AB - 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.
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U2 - 10.1021/bi2012004
DO - 10.1021/bi2012004
M3 - Article
C2 - 21985608
AN - SCOPUS:80755122888
SN - 0006-2960
VL - 50
SP - 9886
EP - 9900
JO - Biochemistry
JF - Biochemistry
IS - 45
ER -