This study identified and characterized two independent enzymic systems for carbamoyl phosphate biosynthesis in Saccharomyces cerevisiae using genetic and biochemical approaches. The researchers isolated mutants impaired in carbamoyl phosphate synthesis and determined that both systems employ glutamine as the nitrogen donor, contrary to the carbamoyl phosphokinase pathway. One system is arginine-pathway-specific and repressed by arginine; the other is pyrimidine-pathway-specific and subject to feedback inhibition by UTP. Mutations mapped to three unlinked loci—two (cpa₁ and cpa₂) affecting the arginine system and one (cpu) affecting the pyrimidine system. Unlike Neurospora, carbamoyl phosphate in yeast freely transfers between pathways. Complementation studies revealed that cpa₁ and cpa₂ mutations define distinct genes but can partially restore activity in vitro when combined, suggesting they encode different components of the same enzymic complex. These findings establish the glutamine-dependent carbamoyl phosphate synthetase as the physiologically relevant enzyme in yeast, supported by regulatory properties reflecting each system's metabolic role.

Key findings

• Two independent glutamine-dependent carbamoyl phosphate synthetases exist in S. cerevisiae—one specific for arginine biosynthesis (repressed by arginine) and one for pyrimidine biosynthesis (inhibited by UTP).
• Mutations in three unlinked loci control carbamoyl phosphate synthesis: cpa₁ and cpa₂ affect the arginine-specific system, while cpu affects the pyrimidine-specific system.
• Carbamoyl phosphate synthesized by either pathway is freely available to both pathways in yeast, unlike the channeled system in Neurospora.
• The arginine-specific carbamoyl phosphate synthetase is strongly repressed by arginine (to 10% activity), while the pyrimidine-specific system shows only weak repression by uracil but strong feedback inhibition by UTP.
• cpa₁ and cpa₂ mutations in separate genes can complement each other in diploids and partially restore activity when cell-free extracts are combined, indicating distinct enzymic components.