Studies on channeling of carbamoyl-phosphate in the multienzyme complex that initiates pyrimidine biosynthesis in rat ascites hepatoma cells.

Abstract

Carbamoyl-phosphate synthetase II of higher animals, the first enzyme of de novo pyrimidine biosynthesis, forms a multienzyme complex with aspartate carbamoyltransferase and dihydroorotase, the second and third enzymes of the pathway. The hypothesis that the complex serves to channel carbamoyl-phosphate, synthesized by the first enzyme of the complex, to the second enzyme was tested using a highly purified complex preparation from Yoshida ascites hepatoma cells (AH 13). Experimentally, aspartate carbamoyltransferase in the complex was allowed to compete with exogenously added ornithine carbamoyltransferase, another carbamoyl-phosphate-utilizing enzyme, for carbamoyl-phosphate which was either synthesized endogenously or added exogenously. The ratios of amounts of the two enzymic products, carbamoyl-aspartate and citrulline, were compared. In the absence of enzyme stabilizers dimethyl sulfoxide or glycerol, a slight channeling of the intermediate in the complex was observed. The further addition of 5-phosphoribosyl 1-pyrophosphate, MgUTP (positive and negative allosteric effectors of carbamoyl-phosphate synthetase II), 30% (v/v) dimethyl sulfoxide or 30% (w/v) glycerol did not affect the extent of channeling. It was slightly increased in the presence of 7.5% (v/v) dimethyl sulfoxide plus 2.5% (w/v) glycerol. Any shift of the assay temperature, pH or concentration of MgATP or of the enzyme complex resulted in little further increase in the extent of channeling. Even when a larger amount of the enzyme complex was used to approximate physiological conditions, there was no increase in the extent of channeling either without or with allosteric effectors. MgUTP even abolished channeling under these conditions. These results indicate that carbamoyl-phosphate can be channeled in the multienzyme complex of AH 13 cells, but the extent of channeling is very small, contrary to expectation.