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 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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2014

Author dc:creator

• Welches, William Robert

• Chemistry, Biochemistry

Language dc:language

eng

Identifier

(UMI)AAI8026617

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/67401