title: Impact of the C-terminal loop of histidine triad nucleotide binding protein1 (Hint1) on substrate specificity.
abstract: Although highly sequence similar, human histidine triad nucleotide binding protein (hHint1) and E. coli hinT (echinT) exhibit significant differences in their phosphoramidase substrate specificity and lysyl-adenylate hydrolytic activity. Observing that the C termini of each enzyme are highly dissimilar, we created two chimeric Hint's: one in which the C terminus of hHint1 was replaced with the C terminus of echinT (Hs/ec) and the other in which the C terminus of echinT was replaced with the C terminus of hHint1 (ec/Hs). The Hs/ec chimera exhibited nearly identical specificity constants (kcat/Km) to those found for echinT, whereas the specificity constants of the ec/Hs chimera were found to approximate those for hHint1. In particular, as observed for echinT, the Hs/ec chimera does not exhibit a preference for phosphoramidates containing d- or l- tryptophan, while the ec/Hs chimera adopts the human enzyme preference for the l configuration. In addition, the studies with each chimera revealed that differences in the ability of hHint1 and echinT to hydrolyze lysyl-AMP generated by either E. coli or human lysyl-tRNA synthetase were partially transferable by C-terminal loop exchange. Hence, our results support the critical role of the C-terminal loop of human and E. coli Hint1 on governing substrate specificity.
authors.affiliation: Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Tsui-Fen
authors.initials: TF
authors.lastname: Chou
authors.forename: Yuk Y
authors.initials: YY
authors.lastname: Sham
authors.forename: Carston R
authors.initials: CR
authors.lastname: Wagner
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000737:chemistry; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; C063306:HINT1 protein, human; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D017766:Acid Anhydride Hydrolases; C525148:HinT protein, E coli