Mutations that cause threonine sensitivity identify catalytic and regulatory regions of the aspartate kinase of Saccharomyces cerevisiae

The HOM3 gene of Saccharomyces cerevisiae encodes aspartate kinase, which c atalyses the first step in the branched pathway leading to the synthesis of threonine and methionine from aspartate. Regulation of the carbon flow int o this pathway takes place mainly by feedback inhibition of this enzyme by threonine. We have isolated and characterized three HOM3 mutants that show growth inhibition by threonine due to a severe, threonine-induced reduction of the carbon how into the aspartate pathway, leading to methionine limita tion. One of the mutants has an aspartate kinase which is 30-fold more stro ngly inhibited by threonine than the wild-type enzyme. The predicted amino acid substitution in this mutant, A406T, is located in a region associated with the modulation of the enzymatic activity. The other two mutants carry an aspartate kinase with reduced affinity for its substrates, aspartate and ATP. The corresponding amino acid substitutions, K26I and G25D, affect res idues located in the vicinity of a highly conserved lysine-phenylalanine-gl ycine-glycine (KFGG) stretch present in the N-terminal part of the aspartat e kinase, to which no function has so far been assigned. We suggest that th is region is involved in substrate binding. Mutagenesis of a HOM3 region ce ntred in the KFGG-coding triplets generated alleles that determine threonin e sensitivity or auxotrophy for threonine and methionine, but not a phenoty pe associated with a feedback-resistant aspartate kinase, indicating that t his region is not involved in the allosteric response of the enzyme. Copyri ght (C) 1999 John Wiley & Sons, Ltd.