Acetohydroxy Acid Synthetase with a p H Optimum of 7.5 from Neurospora crassa Mitochondria: Characterization and Partial Purification

Abstract

An acetohydroxy acid synthetase (AAS) has been found associated with the mitochondrial fraction of wild-type Neurospora crassa . It has a p H optimum of 7.5 and is presumed to be homologous to the p H 8.0 AAS that synthesizes the valine and isoleucine precursors in bacteria and yeast. The enzyme was characterized and purified 30- to 60-fold. The AAS activity of intact mitochondria requires thiamine pyrophosphate (TPP), Mn 2+ or Mg 2+ , and flavine adenine dinucleotide (FAD), and is sensitive to end product inhibition by l -valine. This inhibition is p H-dependent and noncompetitive with respect to pyruvate. Activity is slightly repressed during exponential growth in the presence of valine, isoleucine, and leucine. Extraction of the AAS from the mitochondria has a profound influence on the following properties: p H optimum, sensitivity to l -valine, response to FAD, binding of TPP, apparent K m , and stability at 0 to 4 C. The catalytic properties of the partially purified enzyme are described. Two forms of the partially purified AAS can be isolated from preparative Sephadex G-200 chromatographic columns. Both forms are electrophoretically and antigenically similar but one form has an estimated molecular weight of 110,000 to 120,000 whereas the predominant form is a much larger and more buoyant molecule.