Active center studies on bacterial luciferase: modification of the enzyme with 2,4-dinitrofluorobenzene.
Bacterial luciferase catalyzes the mixed-function oxidation of a long-chain saturated aldehyde and FMNH2 to yield the carboxylic acid, FMN, and blue-green light. The enzyme was inactivated by 2,4-dinitrofluorobenzene (FDNB) with an observed second-order rate constant (k2(obsd) of 157 M-1 min-1 at pH 7.0, 25 degrees C; activity was not recovered upon treatment with 2-mercaptoethanol (thiolysis), demonstrating that the inactivation was the result of reaction with one or more amino groups. The dinitrophenyl (DNP) moiety was incorporated into the alpha subunit approximately twice as fast as it was incorporated into the beta subunit; the rate of inactivation was nearly identical with the rate of incorporation into the alpha beta dimer. The incorporation of 1 mol of DNP/alpha beta resulted in complete inactivation, demonstrating that modification of either alpha or beta is sufficient to cause inactivation. Incorporation of DNP into one subunit appeared to either block or decrease the rate of incorporation of DNP into the other subunit. The luciferase was protected from inactivation by binding of long-chain aldehydes or FMN. Following modification by FDNB, the enzyme had lost measurable FMNH2 binding. The apparent pKa of the amino groups, determined by analysis of the pH dependence of the inactivation reaction, was 9.4. This value is too high to allow correlation with the pH-activity profile of the enzyme [Nicoli, M. Z., Meighen, E. A., & Hastings, J. W. (1974) J. Biol. Chem. 249, 2385-2392]. The catalytic function, if any, for the reactive amino groups remains unknown.
D001665:Binding Sites; D004139:Dinitrofluorobenzene / Q000494:pharmacology*; D005486:Flavin Mononucleotide; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism*; D046911:Macromolecular Substances; D009578:Nitrobenzenes / Q000494:pharmacology*; D010084:Oxidation-Reduction; D011485:Protein Binding; D014316:Tritium; D014733:Vibrio / Q000201:enzymology*; D014737:Vibrionaceae / Q000201:enzymology*
D046911:Macromolecular Substances; D009578:Nitrobenzenes; D014316:Tritium; D005486:Flavin Mononucleotide; D004139:Dinitrofluorobenzene; D008156:Luciferases