title: Regulation of yeast acetohydroxyacid synthase by valine and ATP.
abstract: The first step in the common pathway for the biosynthesis of branched-chain amino acids is catalysed by acetohydroxyacid synthase (AHAS; EC 4.1.3.18). The enzyme is found in plants, fungi and bacteria, and is regulated by controls on transcription and translation, and by allosteric modulation of catalytic activity. It has long been known that the bacterial enzyme is composed of two types of subunit, and a similar arrangement has been found recently for the yeast and plant enzymes. One type of subunit contains the catalytic machinery, whereas the other has a regulatory function. Previously, we have shown [Pang and Duggleby (1999) Biochemistry 38, 5222--5231] that yeast AHAS can be reconstituted from its separately purified subunits. The reconstituted enzyme is inhibited by valine, and ATP reverses this inhibition. In the present work, we further characterize the structure and the regulatory properties of reconstituted yeast AHAS. High phosphate concentrations are required for reconstitution and it is shown that these conditions are necessary for physical association between the catalytic and regulatory subunits. It is demonstrated by CD spectral changes that ATP binds to the regulatory subunit alone, most probably as MgATP. Neither valine nor MgATP causes dissociation of the regulatory subunit from the catalytic subunit. The specificity of valine inhibition and MgATP activation are examined and it is found that the only effective analogue of either regulator of those tested is the non-hydrolysable ATP mimic, adenosine 5'-[beta,gamma-imido]triphosphate. The kinetics of regulation are studied in detail and it is shown that the activation by MgATP depends on the valine concentration in a complex manner that is consistent with a proposed quantitative model.
authors.affiliation: Centre for Protein Structure, Function and Engineering, Department of Biochemistry and Molecular Biology, The University of Queensland, Brisbane QLD 4072, Australia.
authors.forename: S S
authors.initials: SS
authors.lastname: Pang
authors.forename: R G
authors.initials: RG
authors.lastname: Duggleby
mesh_terms: D000095:Acetolactate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D017360:Arabidopsis / Q000201:enzymology; D001667:Binding, Competitive; D002384:Catalysis; D003429:Cross Reactions; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014633:Valine / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D000255:Adenosine Triphosphate; D000095:Acetolactate Synthase; D014633:Valine