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N-terminal histidines are responsible for the decrease in luciferase activity at pH 8
Liyun Liu*; DL Robertson; JW Hastings
Dept. of Molecular & Cellular Biology, 16 Divinity Avenue, Cambridge, MA 02138, USA *(liyunliu@fas.Harvard.edu)
Gonyaulax luciferase is a single chain (~137 kDa) polypeptide, whose activity is highly pH-dependent; flashing in vivo is postulated to be triggered by a pH decrease in scintillons. The protein has three homologous domains, each with activity. In previous work the activity of the full length native enzyme was found to be maximal at pH 6.3 and near zero at pH 8, while the activity of proteolytic fragments remained relatively high at pH 8. While the decrease in activity at alkaline pH also occurred with recombinant peptides of complete single domains, the removal of ~ 60 N-terminal amino acids from each domain increased the pH 8/ 6.3 activity ratio. Three intramolecularly conserved histidine residues are located in the N-terminal repeat regions; in this work these were replaced by alanines in the second of the three domains. The resulting peptides have specific activities at pH 6.3 similar to the unaltered peptides, but their activities at pH 8 are greater. The activity at pH 8 of the peptide with both of the first two histidines substituted is even greater than that at pH 6.3. The results support the hypothesis that these histidine residues are responsible for a loss in luciferase activity at alkaline pH in the native molecule.
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