The role of active site residue arginine 218 in firefly luciferase bioluminescence.
Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate firefly luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We had previously developed a working model of the luciferase active site based on the X-ray structure of the enzyme without bound substrates. In our model, the side chain guanidinium group of Arg218 appears to be located in close proximity to the substrate's hydroxyl group at the bottom of the luciferin binding pocket. A similar role for Arg337 also has been proposed. We report here the construction, purification, and characterization of mutant luciferases R218A, R218Q, R218K, R337Q, and R337K. Alteration of the Arg218 side chain produced enzymes with 15-20-fold increases in the Km values for luciferin. The contrasting near-normal Km values for luciferin determined with the Arg337 enzymes support our proposal that Arg218 (and not Arg337) is an essential luciferin binding site residue. Bioluminescence emission studies indicated that in the absence of a positively charged group at position 218, red bioluminescence was produced. Based on this result and those of additional fluorescence experiments, we speculate that Arg218 maintains the polarity and rigidity of the emitter binding site necessary for the normal yellow-green emission of P. pyralis luciferase. The findings reported here 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
Magyar
R A
RA
Murtiashaw
M H
MH
Portier
N C
NC
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D001517:Coleoptera / Q000201:enzymology*; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity / Q000235:genetics
D011994:Recombinant Proteins; D001120:Arginine; D008156:Luciferases