title: Effect of a domain-spanning disulfide on aminoacyl-tRNA synthetase activity.
abstract: Enzymes regulated by allostery undergo conformational rearrangement upon binding effector molecules. For modular proteins, a flexible interface may mediate reorientation of the protein domains and transmit binding events to activate catalysis at a distance. Aminoacyl-tRNA synthetases (aaRSs) that use tRNA anticodons as identity elements can be considered allosteric enzymes in which aminoacylation of the tRNA acceptor stem is enhanced upon anticodon binding. We reasoned that anticodon-triggered conformational change might be restricted upon introduction of a disulfide linkage near the core of an aaRS. Here we show that a double cysteine mutation engineered at the Escherichia coli MetRS domain interface spontaneously generates a disulfide linkage. This disulfide clamp has no effect on methionyl adenylate formation but reduces the level of tRNA(Met) aminoacylation approximately 2-fold. Activity is restored upon chemical reduction of the disulfide, demonstrating that E. coli MetRS requires a flexible interface domain for full catalytic efficiency.
authors.affiliation: Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109, USA.
authors.forename: Papri
authors.initials: P
authors.lastname: Banerjee
authors.forename: M Bryan
authors.initials: MB
authors.lastname: Warf
authors.forename: Rebecca
authors.initials: R
authors.lastname: Alexander
mesh_terms: D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D008718:Methionine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism
chemical_list: D004220:Disulfides; D012346:RNA, Transfer, Amino Acyl; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase