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The role of firefly luciferase C-terminal domain in efficient coupling of adenylation and oxidative steps
Authors:Ayabe Keiichi  Zako Tamotsu  Ueda Hiroshi
Institution:Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Abstract:The N-terminal domain (N-domain) of the firefly luciferase from Photinus pyraris has weak luminescence activity, and shows a unique light emitting profile with very long rise time of more than several minutes. Through a sensitive assay of the reaction intermediate luciferyl-adenylate (LH2-AMP), we found that the slow increase in the N-domain luminescence faithfully reflected the concentration of dissociated LH2-AMP. No such correlation was observed for wild-type or mutant enzymes with short rise time, except one with longer rise time. The results suggest that the C-terminal domain plays an indispensable role in efficiently coupling adenylation and oxidative steps.
Keywords:C-domain  C-terminal (441-550 aa) domain of Photinus pyralis luciferase  CoA  coenzyme A  GST  glutathione S-transferase  LH2  d-luciferin" target="_blank">d-luciferin  Luc  firefly luciferase  N-domain  N-terminal (1-440 aa) domain of Photinus pyralis luciferase  PPi  pyrophosphate  Ppy WT  recombinant Photinus pyralis luciferase containing the additional N-terminal peptide GPLGS
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