The role of lysine 529, a conserved residue of the acyl-adenylate-forming enzyme superfamily, in firefly luciferase.
Firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen in a two-step process. The enzyme first catalyzes the adenylation of the carboxylate substrate luciferin with Mg-ATP followed by the oxidation of the acyl-adenylate to the light-emitting oxyluciferin product. The beetle luciferases are members of a large family of nonbioluminescent proteins that catalyze reactions of ATP with carboxylate substrates to form acyl-adenylates. Formation of the luciferase-luciferyl-AMP complex is a specific example of the chemistry common to this enzyme family. Site-directed mutants at positions Lys529, Thr343, and His245 were studied to determine the effects of the amino acid changes at these positions on the individual luciferase-catalyzed adenylation and oxidation reactions. The results suggest that Lys529 is a critical residue for effective substrate orientation and that it provides favorable polar interactions important for transition state stabilization leading to efficient adenylate production. These findings as well as those with the Thr343 and His245 mutants are interpreted in the context of the firefly luciferase X-ray structures and computational-based models of the active site.
Branchini
B R
BR
Department of Chemistry, Connecticut College, New London, Connecticut 06320, USA. brbra@conncoll.edu
Murtiashaw
M H
MH
Magyar
R A
RA
Anderson
S M
SM
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001517:Coleoptera / Q000201:enzymology*; D049454:Firefly Luciferin / Q000096:biosynthesis; D006860:Hydrogen Bonding; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics; D008163:Luminescent Measurements; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
D000249:Adenosine Monophosphate; D049454:Firefly Luciferin; D000255:Adenosine Triphosphate; D008156:Luciferases; D008239:Lysine