RECONSTITUTION AND CHARACTERIZATION OF THE POLYNUCLEAR IRON-SULFUR CLUSTER IN PYRUVATE FORMATE-LYASE-ACTIVATING ENZYME - MOLECULAR-PROPERTIES OF THE HOLOENZYME FORM

Citation
R. Kulzer et al., RECONSTITUTION AND CHARACTERIZATION OF THE POLYNUCLEAR IRON-SULFUR CLUSTER IN PYRUVATE FORMATE-LYASE-ACTIVATING ENZYME - MOLECULAR-PROPERTIES OF THE HOLOENZYME FORM, The Journal of biological chemistry, 273(9), 1998, pp. 4897-4903
Citations number
28
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
ISSN journal
0021-9258
Volume
273
Issue
9
Year of publication
1998
Pages
4897 - 4903
Database
ISI
SICI code
0021-9258(1998)273:9<4897:RACOTP>2.0.ZU;2-2
Abstract
The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is generated by the S-adenosyl methionine-dependent pyruvate formate-lyase-activating enzyme (PFL act ivase), A 5'-deoxyadenosyl radical intermediate produced by the activa se has been suggested as the species that abstracts the pro-S hydrogen of the glycine 734 residue in PFL (Frey, M., Rothe, M., Wagner, A. F. V., and Knappe, J. (1994) J. Biol. Chem. 269, 12432-12437). To enable mechanistic investigations of this system we have worked out a conven ient large scale preparation of functionally competent PFL activase fr om its apoform, The previously inferred metallic cofactor was identifi ed as redox-interconvertible polynuclear iron-sulfur cluster, most pro bably of the [4Fe-4S] type, according to W-visible and EPR spectroscop ic information, Cys --> Ser replacements by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to be essential to yield active holoenzyme, Gel filtration chromatography showed a monomeric structure (28 kDa) for both the apoenzyme and holoenzyme form, The iron-sulfur cluster complement proved to be a prerequisite for effective binding o f adenosylmethionine, which induces a characteristic shift of the EPR signal shape of the reduced enzyme form ([4Fe-4S](+)) from axial to rh ombic symmetry.