Regulation of yeast acetohydroxyacid synthase by valine and ATP

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 con trols on transcription and translation, and by allosteric modulation of cat alytic activity. It has long been known that the bacterial enzyme is compos ed of two types of subunit, and a similar arrangement has been found recent ly for the yeast and plant enzymes. One type of subunit contains the cataly tic 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, reco nstituted enzyme is inhibited by valine, and ATP reverses this inhibition. In the present work, we further characterize the structure and the regulato ry properties of reconstituted yeast AHAS. High phosphate concentrations ar e required for reconstitution and it is shown that these conditions are nec essary for physical association between the catalytic and regulatory subuni ts. It is demonstrated by CD spectral changes that ATP binds to the regulat ory subunit alone, most probably as MgATP. Neither valine nor MgATP causes dissociation of the regulatory subunit from the catalytic subunit. The spec ificity of valine inhibition and MgATP activation are examined and it is fo und that the only effective analogue of either regulator of those tested is the non-hydrolysable ATP mimic, adenosine 5 '-[beta,gamma -imido]triphosph ate. 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 ma nner that is consistent with a proposed quantitative model.