Impact of the C-terminal loop of histidine triad nucleotide binding protein1 (Hint1) on substrate specificity.
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.
Chou
Tsui-Fen
TF
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Sham
Yuk Y
YY
Wagner
Carston R
CR
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
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