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|Title:||Active Center Studies on Bacterial Luciferase: Modification of the Enzyme With 2,4-Dinitrofluorobenzene|
|Author(s):||Welches, William Robert|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Bacterial luciferase catalyzes the mixed-function oxidation of a long-chain saturated aldehyde and FMNH(,2) to yield the carboxylic acid, FMN and blue-green light. The enzyme is inactivated by 2,4-dinitrofluorobenzene (FDNB) with an observed second-order rate constant (k(,2(obs))) of 157 M('-1) min('-1) at pH 7.0, 25(DEGREES); activity is not recovered upon treatment with 2-mercaptoethanol (thiolysis), demonstrating that the inactivation is the result of reaction with one or more amino groups. The dinitrophenyl moiety is incorporated into the (alpha) subunit approximately twice as fast as it is incorporated into the (beta) subunit; the rate of inactivation is nearly identical to the rate of incorporation into the (alpha)(beta) dimer. The incorporation of 1 mole DNP/(alpha)(beta) results in complete inactivation. Modification of either (alpha) or (beta) is sufficient to cause inactivation. Analysis of acid hydrolysates of modified enzyme showed that the most likely sites of modification are the NH(,2)-terminal methionyl residues of the two subunits to form the acid-labile DNP-methionine rather than the acid-stable (epsilon)-DNP-lysine.
The luciferase is protected from inactivation by binding of long-chain aldehydes or FMN. Following modification by FDNB, the enzyme has lost measurable FMNH(,2) binding.
The apparent pK(,a) 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., and Hastings, J. W. (1974) J. Biol. Chem. 249, 2385-2392). The catalytic function (if any) for the reactive amino groups remains unknown.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1980.
|Date Available in IDEALS:||2014-12-14|