Metabolic compartmentation at the molecular level: The function of a multienzyme aggregate in the pyrimidine pathway of yeast

Abstract

In yeast, the pyrimidine-specific carbamyl phosphate synthetase and aspartate transcarbamylase exist as an aggregate, together with a regulatory site. The hypothesis, originally proposed by Davis, that such complexes serve to channel the product of the first enzyme ( i.e. carbamyl phosphate which is common to both the pyrimidine pathway and the arginine pathway) into the pathway for which it was produced has been subjected to two experimental tests. In both types of experiment, controls were run in which the two enzymes were separate. When the activities were separate, cold carbamyl phosphate in the assay mixture could easily dilute label from [ 14C]bicarbonate subsequently recovered in the form of carbamyl aspartate, the product of aspartate transcarbamylase. Where the activities were associated in the aggregate, the label recovered in the product could not be diluted by the presence of moderate concentrations of cold exogenous carbamyl phosphate. When teh activities were separate, purified bacterial ornithine transcarbamylase could compete for the carbamyl phosphate formed by the action of carbamyl phosphate synthetase on equal terms with the aspartate transcarbamylase; the proportion recovered as either citrulline or carbamyl aspartate was determined by the ratio of activity ornithine transcarbamylase: aspartate transcarbamylase. Where the carbamyl phosphate synthetase and the aspartate transcarbamylase activities were associated in the complex, most of the product of the first reaction was not free to become converted to citrulline but was channeled preferentially into carbamyl aspartate. Both types of experiment confirm the channeling hypothesis.