Tryptophan biosynthesis in Saccharomyces cerevisiae: control of the flux through the pathway

Abstract

Enzyme derepression and feedback inhibition of the first enzyme are the regulatory mechanisms demonstrated for the tryptophan pathway in S. cerevisiae. The relative contributions of the two mechanisms to the control of the flux through the pathway in vivo were analyzed by measuring feedback inhibition of anthranilate synthase in vivo, determining the effect of regulatory mutations on the level of the tryptophan pool and the flux through the pathway, and varying the gene dose of individual enzymes of the pathway at the tetraploid level. It is concluded that the flux through the pathway is adjusted to the rate of protein synthesis by means of feedback inhibition of the first enzyme by the end product, tryptophan. The synthesis of the tryptophan enzymes could not be repressed below a basal level by tryptophan supplementation of the media. The enzymes are present in excess. Increasing or lowering the concentration of individual enzymes had no noticeable influence on the overall flux to tryptophan. The uninhibited capacity of the pathway could be observed both upon relieving feedback inhibition by tryptophan limitation and in feedback-insensitive mutants. It exceeded the rate of consumption of the amino acid on minimal medium by a factor of three. Tryptophan limitation caused derepression of four of the five tryptophan enzymes and, as a consequence, led to a further increase in the capacity of the pathway. However, because of the large reserve capacity of the 'repressed' pathway, tryptophan limitation could not be imposed on wild-type cells without resorting to the use of analogs. The results suggest that derepression does not serve as an instrument for the specific regulation of the flux through the tryptophan pathway.