title: Luminescent and substrate binding activities of firefly luciferase N-terminal domain.
abstract: Firefly luciferase catalyzes highly efficient emission of light from the substrates luciferin, Mg-ATP, and oxygen. A number of amino acid residues are identified to be important for the luminescent activity, and almost all the key residues are thought to be located in the N-terminal domain (1-437), except one in the C-terminal domain, Lys529, which is thought to be critical for efficient substrate orientation. Here we show that the purified N-terminal domain still binds to the substrates luciferin and ATP with reduced affinity, and retains luminescent activity of up to 0.03% of the wild-type enzyme (WT), indicating that all the essential residues for the activity are located in the N-terminal domain. Also found is low luminescence enhancement by coenzyme A (CoA), which implies a lower product inhibition than in the WT enzyme. These findings have interesting implications for the light emission reaction mechanism of the enzyme, such as reaction intermediates, product inhibition, and the role of the C-terminal domain.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656, Japan.
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Zako
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ayabe
authors.forename: Takahide
authors.initials: T
authors.lastname: Aburatani
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kitayama
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D003065:Coenzyme A / Q000737:chemistry; D001517:Coleoptera / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049454:Firefly Luciferin / Q000737:chemistry / Q000378:metabolism; D008156:Luciferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D008274:Magnesium / Q000737:chemistry; D011485:Protein Binding; D013053:Spectrophotometry
chemical_list: D049454:Firefly Luciferin; D000255:Adenosine Triphosphate; D008156:Luciferases; D008274:Magnesium; D003065:Coenzyme A