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Site-directed mutagenesis of histidine 245 in firefly luciferase: a proposed model of the active site.
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, and a structure-based interpretation of the role of 244HHGF247 in firefly biolumscence is provided.
The role of lysine 529, a conserved residue of the acyl-adenylate-forming enzyme superfamily, in firefly luciferase.
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.
Mutagenesis evidence that the partial reactions of firefly bioluminescence are catalyzed by different conformations of the luciferase C-terminal domain.
Evidence is provided that Lys443 and Lys529, located on opposite sides of the C-terminal domain and conserved in all firefly luciferases, are each essential for only one of the partial reactions of firefly bioluminescence, supporting the proposal that the superfamily enzymes may adopt two different conformations to catalyze the two half-reactions.
Crystal structure of firefly luciferase in a second catalytic conformation supports a domain alternation mechanism.
The structure of a cross-linked luciferase enzyme that is trapped in the second conformation is determined, which supports the role of the second catalytic conformation and provides insights into the biochemical mechanism of theLuciferase oxidative step.
A mutagenesis study of the putative luciferin binding site residues of firefly luciferase.
An interpretation of the roles of the mutated residues in substrate binding and bioluminescence color determination is reported, and the foundation for future experiments designed to alter the substrate specificity of firefly luciferase is provided.
An alternative mechanism of bioluminescence color determination in firefly luciferase.
The results of mutagenesis studies designed to determine the basis of the observed differences in bioluminescence color with the analogue adenylate are reported, which show that luciferase modulates emission color by controlling the resonance-based charge delocalization of the anionic keto form of the oxyluciferin excited state.
Mutation of a Protease-sensitive Region in Firefly Luciferase Alters Light Emission Properties*
Results provide a new form of the enzyme that affords a more versatile reporter system and indicate the involvement of this region in ATP binding, as indicated in EC 1.13.12.