Regulation of Isoleucine-Valine Biosynthesis in Saccharomyces cerevisiae

Abstract

An isoleucine requiring mutant (M6) of Saccharomyces cerevisiae was found to have an altered threonine deaminase. The mutation maps in the is1 locus which corresponds to a threonine deaminase deficiency in other isoleucine requiring mutants. Both the molecular weight and the absolute number of sites for the ligands remain the same in the wild type and the mutant enzyme. The following catalytic and regulatory properties are modified in the mutant enzyme: 1 Affinity towards threonine is 4 to 6 fold lower. Also, in contrast with the wild type, coperativity between substrate molecules is maintained at pH 7.5. 2 Affinity towards the substrate analogue (competitive inhibitor), allothreonine, is 2 to 3 fold lower. 3 Affinity towards a feedback inhibitor, isoleucine, is increased by 5 to 6 fold. 4 Valine remains active as a positive effector (suppressing the cooperativity between substrate molecules) but a 20 fold higher concentration is required for a 50% maximal effect. On the other hand, at high concentrations, valine is inhibitory. The apparent Ki, at substrate saturation, is 36 mM. The changes in kinetic properties of the mutant enzyme, which occurred for each individual ligand in the most unfavorable direction, can explain its complete inefficiency in vivo, and account for the isoleucine requirement of the mutant strain. Growth studies have shown that the imparied enzyme can, in vivo, utilize exogenous threonine and be activated by exogenously supplied valine. These results have been interpreted as an indication that endogenous threonine and valine concentrations were the factors responsible for the mutant inability to grow in minimal medium. In addition to explaining the physiological behavior of the mutant M 6, the present study contributes information on the interdependence between various ligand sites on the yeast threonine deaminase.