Subunit association in acetohydroxy acid synthase isozyme III.
Acetohydroxy acid synthase isozyme III (AHAS III) from Escherichia coli is composed of large and small subunits (encoded by the genes ilvI and ilvH) in an alpha 2 beta 2 structure. The large (61-kDa) subunit apparently contains the catalytic machinery of the enzyme, while the small (17-kDa) subunit is required for specific stabilization of the active conformation of the large subunit as well as for valine sensitivity. The interaction between subunits has been studied by using purified enzyme and extracts containing subcloned subunits. The association between large and small subunits is reversible, with a dissociation constant sufficiently high to have important experimental consequences: the activity of the enzyme shows a concentration dependence curve which is concave upward, and this dependence becomes linear upon the addition of excess large or small subunits. We estimate that at a concentration of 10(-7) M for each subunit (7 micrograms of enzyme ml-1), the large subunits are only half associated as the I2H2 active holoenzyme. This dissociation constant is high enough to cause underestimation of the activity of AHAS III in bacterial extracts. The true activity of this isozyme in extracts is observed in the presence of excess small subunits, which maintain the enzyme in its associated form. Reexamination of an E. coli K-12 ilvBN+ ilvIH+ strain grown in glucose indicates that AHAS III is the major isozyme expressed. As an excess of small subunits does not influence the apparent Ki for valine inhibition of the purified enzyme, it is likely that valine binds to and inhibits I2H2 rather than inducing dissociation. AHAS I and II seem to show a much lower tendency to dissociate than does AHAS III.
Sella
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C
Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
Weinstock
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O
Barak
Z
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Chipman
D M
DM
D000095:Acetolactate Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D007527:Isoenzymes; D000095:Acetolactate Synthase