Kinetic destabilization of the hydroperoxy flavin intermediate by site-directed modification of the reactive thiol in bacterial luciferase

Husam M. Abu-Soud, A. Clay Clark, Wilson A. Francisco, Thomas O. Baldwin, Frank M. Raushel

Research output: Contribution to journalArticle

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Abstract

Bacterial luciferase catalyzes the formation of visible light, FMN, and a carboxylic acid from FMNH2, O2, and the corresponding aldehyde. The reactive cysteinyl residue at position 106 of the a subunit has been replaced by serine, alanine, and valine by site-directed mutagenesis (Baldwin, T. O., Chen, L. H., Chlumsky, L. J., Devine, J. H., and Ziegler, M. M. (1989) J. Biolumin. Chemilumin. 4, 40-48) and the kinetics of the reaction catalyzed by each mutant protein measured by stopped-flow spectrophotometry at pH 7 and 25°C. The time courses for the formation and decay of the various intermediates for the three αC106 mutants have been followed by monitoring the absorbance at 380 and 445 nm and the emission of visible light using n-decanal as the aldehyde substrate. The time courses for these events have been incorporated into a comprehensive kinetic model; 16 individual rate constants have been obtained for this model by numeric simulations of the time courses for the wild-type enzyme and for the three αC106 mutants. The mutants catalyzed the production of visible light demonstrating that the reactive thiol is not involved in the bioluminescence reaction. All three mutants have been found to catalyze the formation of the C4a-hydroperoxy flavin intermediate with rate constants equal to that of the wild-type enzyme. These results are incompatible with those reported by Xi et al. who have suggested that the major pathway for the oxidation of αC106V-bound FMNH2 does not involve the C4a-hydroperoxy flavin as an intermediate (Xi, L., Cho, K.-W., Herndon, M. E., and Tu, S.-C. (1990) J. Biol. Chem. 265, 4200-4203). The rates of decay of the C4a-hydroperoxy flavin intermediate with the mutant enzymes were found to be two orders of magnitude faster than that of the wild-type enzyme. Luciferase has been shown to be inhibited at high levels of aldehyde substrate when the enzyme is assayed by injecting FMNH2 into an aerobic mixture of enzyme and aldehyde. This aldehyde inhibition has been shown to occur by the formation of a dead-end enzyme-aldehyde complex which blocks the binding of FMNH2 to the enzyme; loss of activity is due to the rapid nonenzymatic decomposition of the reduced flavin with molecular oxygen.

Original languageEnglish (US)
Pages (from-to)7699-7706
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number11
StatePublished - Apr 15 1993
Externally publishedYes

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Bacterial Luciferases
Sulfhydryl Compounds
Aldehydes
Kinetics
Enzymes
Light
Rate constants
Bioluminescence
Flavin Mononucleotide
Mutagenesis
4,6-dinitro-o-cresol
Molecular oxygen
Spectrophotometry
Valine
Mutant Proteins
Substrates
Carboxylic Acids
Site-Directed Mutagenesis
Luciferases
Alanine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Abu-Soud, H. M., Clark, A. C., Francisco, W. A., Baldwin, T. O., & Raushel, F. M. (1993). Kinetic destabilization of the hydroperoxy flavin intermediate by site-directed modification of the reactive thiol in bacterial luciferase. Journal of Biological Chemistry, 268(11), 7699-7706.

Kinetic destabilization of the hydroperoxy flavin intermediate by site-directed modification of the reactive thiol in bacterial luciferase. / Abu-Soud, Husam M.; Clark, A. Clay; Francisco, Wilson A.; Baldwin, Thomas O.; Raushel, Frank M.

In: Journal of Biological Chemistry, Vol. 268, No. 11, 15.04.1993, p. 7699-7706.

Research output: Contribution to journalArticle

Abu-Soud, Husam M. ; Clark, A. Clay ; Francisco, Wilson A. ; Baldwin, Thomas O. ; Raushel, Frank M. / Kinetic destabilization of the hydroperoxy flavin intermediate by site-directed modification of the reactive thiol in bacterial luciferase. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 11. pp. 7699-7706.
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abstract = "Bacterial luciferase catalyzes the formation of visible light, FMN, and a carboxylic acid from FMNH2, O2, and the corresponding aldehyde. The reactive cysteinyl residue at position 106 of the a subunit has been replaced by serine, alanine, and valine by site-directed mutagenesis (Baldwin, T. O., Chen, L. H., Chlumsky, L. J., Devine, J. H., and Ziegler, M. M. (1989) J. Biolumin. Chemilumin. 4, 40-48) and the kinetics of the reaction catalyzed by each mutant protein measured by stopped-flow spectrophotometry at pH 7 and 25°C. The time courses for the formation and decay of the various intermediates for the three αC106 mutants have been followed by monitoring the absorbance at 380 and 445 nm and the emission of visible light using n-decanal as the aldehyde substrate. The time courses for these events have been incorporated into a comprehensive kinetic model; 16 individual rate constants have been obtained for this model by numeric simulations of the time courses for the wild-type enzyme and for the three αC106 mutants. The mutants catalyzed the production of visible light demonstrating that the reactive thiol is not involved in the bioluminescence reaction. All three mutants have been found to catalyze the formation of the C4a-hydroperoxy flavin intermediate with rate constants equal to that of the wild-type enzyme. These results are incompatible with those reported by Xi et al. who have suggested that the major pathway for the oxidation of αC106V-bound FMNH2 does not involve the C4a-hydroperoxy flavin as an intermediate (Xi, L., Cho, K.-W., Herndon, M. E., and Tu, S.-C. (1990) J. Biol. Chem. 265, 4200-4203). The rates of decay of the C4a-hydroperoxy flavin intermediate with the mutant enzymes were found to be two orders of magnitude faster than that of the wild-type enzyme. Luciferase has been shown to be inhibited at high levels of aldehyde substrate when the enzyme is assayed by injecting FMNH2 into an aerobic mixture of enzyme and aldehyde. This aldehyde inhibition has been shown to occur by the formation of a dead-end enzyme-aldehyde complex which blocks the binding of FMNH2 to the enzyme; loss of activity is due to the rapid nonenzymatic decomposition of the reduced flavin with molecular oxygen.",
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T1 - Kinetic destabilization of the hydroperoxy flavin intermediate by site-directed modification of the reactive thiol in bacterial luciferase

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AU - Clark, A. Clay

AU - Francisco, Wilson A.

AU - Baldwin, Thomas O.

AU - Raushel, Frank M.

PY - 1993/4/15

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N2 - Bacterial luciferase catalyzes the formation of visible light, FMN, and a carboxylic acid from FMNH2, O2, and the corresponding aldehyde. The reactive cysteinyl residue at position 106 of the a subunit has been replaced by serine, alanine, and valine by site-directed mutagenesis (Baldwin, T. O., Chen, L. H., Chlumsky, L. J., Devine, J. H., and Ziegler, M. M. (1989) J. Biolumin. Chemilumin. 4, 40-48) and the kinetics of the reaction catalyzed by each mutant protein measured by stopped-flow spectrophotometry at pH 7 and 25°C. The time courses for the formation and decay of the various intermediates for the three αC106 mutants have been followed by monitoring the absorbance at 380 and 445 nm and the emission of visible light using n-decanal as the aldehyde substrate. The time courses for these events have been incorporated into a comprehensive kinetic model; 16 individual rate constants have been obtained for this model by numeric simulations of the time courses for the wild-type enzyme and for the three αC106 mutants. The mutants catalyzed the production of visible light demonstrating that the reactive thiol is not involved in the bioluminescence reaction. All three mutants have been found to catalyze the formation of the C4a-hydroperoxy flavin intermediate with rate constants equal to that of the wild-type enzyme. These results are incompatible with those reported by Xi et al. who have suggested that the major pathway for the oxidation of αC106V-bound FMNH2 does not involve the C4a-hydroperoxy flavin as an intermediate (Xi, L., Cho, K.-W., Herndon, M. E., and Tu, S.-C. (1990) J. Biol. Chem. 265, 4200-4203). The rates of decay of the C4a-hydroperoxy flavin intermediate with the mutant enzymes were found to be two orders of magnitude faster than that of the wild-type enzyme. Luciferase has been shown to be inhibited at high levels of aldehyde substrate when the enzyme is assayed by injecting FMNH2 into an aerobic mixture of enzyme and aldehyde. This aldehyde inhibition has been shown to occur by the formation of a dead-end enzyme-aldehyde complex which blocks the binding of FMNH2 to the enzyme; loss of activity is due to the rapid nonenzymatic decomposition of the reduced flavin with molecular oxygen.

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