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a-Isopropylmalate synthetase which catalyzes the first step in leucine biosynthesis, the formation of a-isopropylmalate from acetyl coenzyme A and aketoisovalerate, has been isolated from Neurospora. The enzyme has a broad pH optimum between pH 7 and 8 and requires potassium ions for activity. The Km value of the enzyme at pH 7.5 is 2.45 X 10~5 m for acetyl coenzyme A and 1.0 X 10-5 m for a-ketoisovalerate.Pyruvate and a-ketobutyrate can substitute for aketoisovalerate as substrates but display Km values of 5.8 X 10-3 and 2.1 X 10~3 m, respectively. Leucine and its structural analog 5',5',5'-trifluoro-DL-leucine are effective inhibitors of synthetase function. The inhibition is reversed by the structurally unrelated subinterest in the structure and function of the Neurospora a-isopropylmalate synthetase (referred to hereafter as the synthetase)* 1 stems from the fact that the enzyme exhibits several biologically important features. The synthetase catalyzes the first reaction in the biosynthetic sequence leading to the formation of leucine and is regulated by the end product of the reaction sequence (Umbarger, 1961). In addition, the synthesis of the synthetase is controlled by a leucine-concentration-dependent repression mechanism (S. R. Gross, in preparation). Furthermore, active synthetase can be formed by complementation interactions between differently defective products of leu-4 alleles.

Examination of the culture fluids of several leucine auxotrophs derived from Neurospora crassa revealed that one strain, 33757, accumulated, in addition to p-carboxy-p-hydroxyisocaproate, a second compound which supported the growth of three of four classes of leucine auxotrophs of Salmonella typhimurium. This compound was isolated and identified as a-hydroxy-p-carboxyisocaproate by nuclear magnetic resonance spectrum and by elementary analysis. The compound was converted to a-ketoisocaproate and carbon dioxide by an NAD-linked dehydrogenase. The enzyme was partially purified from extracts of s. tynhimurium by ammonium sulfate precipitation and by chromatography on brushite. The enzyme exhibited a requirement for both mono-and divalent cations for maximal activity.The optimum pH was 9.5. The formation of the dehydrogenase was dependent upon single structural genes in S. typhimurium and N . crassa.

A technique for the selection of induced and spontaneous auxotrophic mutants of Neurospora in relatively high frequency is described. The method takes advantage of the reduced rate of death of germinating conidia of an inositol-requiring strain when a secondary mutation is imposed.

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