Phosphoribosyl pyrophosphate synthetase (PRPS) is an enzyme conserved across all forms of life, tracing back to the last universal common ancestor (LUCA). PRPS catalyzes the rate-limiting step in converting ribose-5-phosphate (R5P) to phosphoribosyl pyrophosphate (PRPP), a crucial precursor in the biosynthesis of nucleotides, amino acids, and lipids. While Bacteria and Archaea species typically express a single PRPS enzyme, the presence of multiple PRPS-encoding genes is a hallmark trait of eukaryotes. Given the well-established evolutionary trajectory from opisthokonts to mammals, supported by relatively complete molecular phylogenetic data, we used mammals in our study that harbor five PRPS homologs, as a model system to investigate the evolutionary origins and functional significance of these multiple homologs. In addition to the three isozymes– PRPS1, PRPS2, and PRPS1L1 (testes-specific) – mammals also possess PRPSAP1 and PRPSAP2, which feature unique insertions in the catalytic flexible (CF) loop (referred to as non-homologous regions (NHRs)) compared to the sequences found in PRPS isozymes. In this project, to ascertain whether these evolutionary conserved mammalian PRPS homologs interact with each other, we tagged PRPS1, PRPS2, PRPSAP1, and PRPSAP2 individually with GFP at their C-termini. Immunoprecipitation followed by LC-MS/MS peptide identification from all revealed that all PRPS homologs assemble into a single enzyme complex in mouse (NIH3T3) cells. As a control, we used Flag-NES (Nuclear Export Signal)-GFP tagged cells given that PRPS enzymes localize in the cytoplasm. Employing isogenic cells representing all viable individual or combinatorial assembly states, we dissect the basic organizational principles of the PRPS enzyme complex and characterize the emergent properties responsible for paralog specialization, including new modes of regulation that govern complex assembly and activity in vivo. Collectively, our study demonstrates how evolution has transformed a single PRPS enzyme into a biochemical complex endowed with novel functional and regulatory features that fine-tune mammalian metabolism.