Site-directed mutagenesis of histidine 245 in firefly luciferase: a proposed model of the active site.
Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We previously reported [Branchini, B. R., Magyar, R. A., Marcantonio, K. M., Newberry, K. J., Stroh, J. G., Hinz, L. K., and Murtiashaw, M. H. (1997) J. Biol. Chem. 272, 19359-19364] that 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a firefly luciferin analogue, was a potent photoinactivation reagent for luciferase. We identified a luciferase peptide 244HHGF247, the degradation of which was directly correlated to the photooxidation process. We report here the construction and purification of wild-type and mutant luciferases H244F, H245F, H245A, and H245D. The results of photoinactivation and kinetic and bioluminescence studies with these proteins are consistent with His245 being the primary functional target of BPTC-catalyzed enzyme inactivation. The possibility that His245 is oxidized to aspartate during the photooxidation reaction was supported by the extremely low specific activity ( approximately 300-fold lower than WT) of the H245D mutant. Using the crystal structures of luciferase without substrates [Conti, E., Franks, N. P., and Brick, P. (1996) Structure 4, 287-298] and the functionally related phenylalanine-activating subunit of gramicidin synthetase 1 [Conti, E., Stachelhaus, T., Marahiel, M. A., and Brick, P. (1997) EMBO J. 16, 4174-4183] as a starting point, we have performed molecular-modeling studies and propose here a model for the luciferase active site with substrates luciferin and Mg-ATP bound. We have used this model to provide a structure-based interpretation of the role of 244HHGF247 in firefly bioluminescence.
Branchini
B R
BR
Department of Chemistry, Connecticut College, New London 06320, USA. brbra@conncoll.edu
Magyar
R A
RA
Murtiashaw
M H
MH
Anderson
S M
SM
Zimmer
M
M
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002264:Carboxylic Acids / Q000378:metabolism; D002384:Catalysis; D001517:Coleoptera; D003198:Computer Simulation; D006639:Histidine / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D008163:Luminescent Measurements; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010782:Photolysis; D017319:Photosensitizing Agents / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity / Q000235:genetics; D013844:Thiazoles / Q000378:metabolism
C115520:2-(4-benzoylphenyl)thiazole-4-carboxylic acid; D002264:Carboxylic Acids; D010446:Peptide Fragments; D017319:Photosensitizing Agents; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; D006639:Histidine; D008156:Luciferases