Active Center Structure and Sequence Studies on Bacterial Luciferase Utilizing the Essential Cysteine, Protease-Labile Region, and Delta Fragment

This study examines the structure of the active center of bacterial luciferase by exploiting the existence of two well-documented structural features of the (alpha) subunit of the enzyme, the essential cysteine and the protease-labile region, which are thought to reside there. Chemical modification of this cysteinyl residue causes slight perturbations, dependent upon the structure of the modifying reagent, in the structure of the protease-labile region as well as loss of enzymatic activity, as does intrasubunit crosslinking induced by the reagent p-azidophenacyl bromide. High concentrations of phosphate ion or the reaction product FMN slightly decrease the reactivity of the essential cysteine. These results and those of limited proteolysis of luciferase modified at this cysteine with ('3)H-N-ethylmaleimide, N-terminal sequence analysis of the resulting fragments, and specific cleavage of (alpha) at the essential thiol caused by modification with 2-nitro-5-thiocyanatobenzoic acid, are consistent with a model which places this residue on an exposed polypeptide loop, which is a portion of the active center, (TURN)100 residues from the amino terminus of (alpha). The protease-labile region appears to be composed of two sections of primary structure, one located on the same polypeptide loop as the essential cysteine (the "slow" area), and the other (TURN)100-130 residues from the carboxy terminus (the "fast" area). Amino acid sequences of peptides derived from the proteolytic fragments of the "(delta)" family are also presented.

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

• Rausch, Steven Kirk

• Chemistry, Biochemistry

Identifier

(UMI)AAI8310002

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/70522