Impact of the C-terminal loop of histidine triad nucleotide binding protein 1 (Hint1) on substrate specificity

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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 hHintl 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 hHintl (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 hHintl 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.

Original languageEnglish (US)
Pages (from-to)13074-13079
Number of pages6
JournalBiochemistry
Volume46
Issue number45
DOIs
StatePublished - Nov 13 2007

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Substrate Specificity
Histidine
Escherichia coli
Carrier Proteins
Nucleotides
Substrates
Lysine-tRNA Ligase
Enzymes
Adenosine Monophosphate
Tryptophan

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Impact of the C-terminal loop of histidine triad nucleotide binding protein 1 (Hint1) on substrate specificity. / Chou, Tsui Fen; Sham, Yuk Y; Wagner, Carston R.

In: Biochemistry, Vol. 46, No. 45, 13.11.2007, p. 13074-13079.

Research output: Contribution to journalArticle

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