title: Effect of iron on activity of soybean multi-subunit acetyl-coenzyme A carboxylase.
abstract: Multi-subunit acetyl-coenzyme A carboxylase (MS-ACCase; EC 6.4.1.2) isolated from soybean chloroplasts is a labile enzyme that loses activity during purification. We found that incubating the chloroplast stromal fraction under anaerobic conditions or in the presence of 5 mM FeSO4 stimulated ACCase (acetyl-CoA-->malonyl-CoA) and carboxyltransferase (malonyl-CoA-->acetyl-CoA) activity. Fe-stimulation of activity was associated with 59Fe binding to a stromal protein fraction. ACCase and carboxyltransferase activities measured in the stromal protein fraction containing bound 59Fe were 2-fold and 6-fold greater, respectively, than the control (stromal fraction not pretreated with FeSO4). Superose 6 gel filtration chromatography indicated 59Fe comigrated with stromal protein of approximately 180 kDa that exhibited carboxyltransferase activity, but lacked ACCase activity. Anion exchange (Mono-Q) chromatography of the Superose 6 fraction yielded a protein peak that was enriched in carboxyltransferase activity and contained protein-bound 59Fe. Denaturing gels of the Mono-Q fraction indicated that the 180-kDa protein was composed of a 56-kDa subunit that was bound by an antibody raised against a synthetic beta-carboxyltransferase (beta-CTase) peptide. Incubation of the Mono-Q carboxyltransferase fraction with increasing concentrations of iron at a fixed substrate concentration resulted in increased initial velocities that fit well to a single rectangular three parameter hyperbola (v=vo+Vmax[FeSO4]/Km+[FeSO4]) consistent with iron functioning as a bound activator of catalysis. UV/Vis spectroscopy of the partially purified fraction before and after iron incubation yielded spectra consistent with a protein-bound metal cluster. These results suggest that the beta-CTase subunit of MS-ACCase in soybean chloroplasts is an iron-containing enzyme, which may in part explain its labile nature.
authors.affiliation: Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA Plant Science Research Unit, USDA-ARS, St. Paul, MN 55108, USA.
authors.forename: David W.
authors.initials: DW
authors.lastname: Plank
authors.forename: Burle G.
authors.initials: BG
authors.lastname: Gengenbach
authors.forename: John W.
authors.initials: JW
authors.lastname: Gronwald