title: Mutations in the regulatory subunit of yeast acetohydroxyacid synthase affect its activation by MgATP.
abstract: Isoleucine, leucine and valine are synthesized via a common pathway in which the first reaction is catalysed by AHAS (acetohydroxyacid synthase; EC 2.2.1.6). This heterotetrameric enzyme is composed of a larger subunit that contains the catalytic machinery and a smaller subunit that plays a regulatory role. The RSU (regulatory subunit) enhances the activity of the CSU (catalytic subunit) and mediates end-product inhibition by one or more of the branched-chain amino acids, usually valine. Fungal AHAS differs from that in other organisms in that the inhibition by valine is reversed by MgATP. The fungal AHAS RSU also differs from that in other organisms in that it contains a sequence insert. We suggest that this insert may form the MgATP-binding site and we have tested this hypothesis by mutating ten highly conserved amino acid residues of the yeast AHAS RSU. The modified subunits were tested for their ability to activate the yeast AHAS CSU, to confer sensitivity to valine inhibition and to mediate reversal of the inhibition by MgATP. All but one of the mutations resulted in substantial changes in the properties of the RSU. Unexpectedly, four of them gave a protein that required MgATP in order for strong stimulation of the CSU and valine inhibition to be observed. A model to explain this result is proposed. Five of the mutations abolished MgATP activation and are suggested to constitute the binding site for this modulator.
authors.affiliation: School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, QL 4072, Australia.
authors.forename: Yu-Ting
authors.initials: YT
authors.lastname: Lee
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Duggleby
mesh_terms: D000095:Acetolactate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000597:Amino Acids, Branched-Chain / Q000096:biosynthesis; D020134:Catalytic Domain; D004789:Enzyme Activation / Q000187:drug effects; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology*
chemical_list: D000597:Amino Acids, Branched-Chain; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D000095:Acetolactate Synthase