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.