Biosynthesis of Branched-Chain Amino Acids in Yeast: Correlation of Biochemical Blocks and Genetic Lesions in Leucine Auxotrophs

Abstract

The three enzymatic steps in the conversion of α-ketoisovalerate to α-ketoisocaproate were examined in wild-type and in leucine auxotrophic stocks of yeast. Procedures for the reliable assay of each of the enzymatic steps in crude extracts were devised. Crude extracts of the prototrophic haploid stock catalyzed all three enzymatic steps. Examination of a series of leucine auxotrophs permitted a correlation between the three enzymatic steps and the genetic lesions affecting 10 different loci. This examination revealed that a single locus ( le -6) affected primarily α-isopropylmalate synthetase, the first step in the pathway. Lesions in six loci ( le -1, le -4, le -5, le -7, le -8, and le -10) lead primarily to a deficiency in the activity of the second enzyme in the pathway, α-isopropylmalate isomerase. Stocks with lesions in three loci ( le -2, le -3, and le -9) were primarily blocked in the third step of the pathway, catalyzed by β-isopropylmalate dehydrogenase. The results with the mutants provide strong evidence that the pathway for leucine biosynthesis proposed by Strassman and his colleagues is the sole significant pathway in yeast.