Phosphoribosyl pyrophosphate synthetase (PRPS) is a highly conserved enzyme that conducts the chokepoint reaction of nucleotide biosynthesis by converting ribose-5-phosphate (R-5-P) to phosphoribosyl pyrophosphate (PRPP). Due to the presence of multiple isoforms and related PRPS-associated proteins in opisthokont species, the precise nature of the PRPS enzyme in cells and tissues remains uncertain. Using proteomics approaches and biochemistry, we demonstrate that these individual components assemble together to form a heterogeneous megadalton complex comprising of PRPS1, PRPS2, and two PRPS associated proteins – PRPSAP1 and PRPSAP2. To validate this and identify other protein/protein complexes that exists in a similar high molecular weight (HMW) range in human (HEK293T) cells, we performed shotgun proteomics on size exclusion chromatography fractions containing HMW proteins/protein complexes. This analysis identified a total of 262 unique proteins, which included ribosomal proteins and CAD confirming enrichment for HMW proteins. This dataset also revealed that among the eight cytosolic enzymes residing in HMW range, two were PRPS isozymes, indicating that the PRPS