Isolation and characterization of subunits of acetohydroxy acid synthase isozyme III and reconstitution of the holoenzyme.
The separately cloned large and small subunits of AHAS isozyme III from Escherichia coli have been isolated and purified. The essentially pure small subunit (17 kDa ilvH product) was obtained by a procedure exploiting its low solubility. The large, catalytic subunit (62 kDa ilvI product) was isolated by standard techniques, to > or = 95% purity. The large subunit has low catalytic activity relative to holoenzyme (approximately 5%) but shows similar substrate specificity and qualitatively similar cofactor dependence and inhibition by a sulfonylurea herbicide. Its activity is insensitive to valine, and the protein does not bind valine. The small subunit binds valine with Kd = 0.2 mM. Reconstitution of the holoenzyme from its subunits leads to a complex with the properties of the native protein, including valine inhibition of activity with Ki = 12 microM. Reconstitution titrations confirm the 1:1 stoichiometry of subunit assembly and a tendency to dissociation (about 50% dissociation near 0.1 microM subunit). Size exclusion HPLC indicates that either subunit alone is largely monomeric, and that assembly of the holoenzyme (two large + two small subunits, 150-160 kDa) requires FAD. On the basis of its homology with pyruvate oxidase and pyruvate decarboxylase, we suggest that the active sites of AHAS III are located at the interface of a dimer of catalytic subunits. Our experiments suggest that such a dimer is not stable except in the presence of the small subunits. The association of valine with sites on the regulatory subunits presumably influences the active sites by an allosteric conformational effect.
Vyazmensky
M
M
Department of Life Science, Ben-Gurion University of the Negev, Israel.
Sella
C
C
Barak
Z
Z
Chipman
D M
DM
D000095:Acetolactate Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D014633:Valine / Q000378:metabolism
D007527:Isoenzymes; D000095:Acetolactate Synthase; D014633:Valine