PROJECT SUMMARY Phosphofructokinase (PFK) is an essential node governing the flux of glucose through glycolysis. It catalyzes the rate limiting step of glycolysis, converting fructose-6-phosphate to fructose-1,6-bisphosphate. Dysfunction of PFK is associated with many diseases including cancer, glycogen storage diseases, heart disease, and many others. As befitting of its role as the gatekeeper of glycolysis, the activity of PFK is tightly regulated. In humans, there are three PFK isoforms: liver (PFKL), platelet (PFKP), and muscle (PFKM), each with unique regulatory properties. Our understanding of these regulatory properties is not complete, mainly because isoform-specific structural information is lacking. Understanding PFK regulation is useful in the design of therapeutics and tools for research. The proposed research will use cryo-EM, mutagenesis, in vitro kinetic assays, biophysical assays, and cell-based assays to better define three aspects of isoform-specific PFK regulation. Firstly, PFKM is unique due to its non-canonical response to pH and ATP. The biophysical basis for the regulation by ATP and pH in PFKM will be assessed using cryo-EM, mutagenesis, and in vitro kinetics assays. Secondly, as structural infor- mation on eukaryotic PFK emerges, differences in the active and inactive conformations have been identified. Specifically, in PFK, the flexible C-terminal tail has been shown to become ordered upon PFK inactivation and loss of the C-terminal tail in PFKL results in altered response to regulation by PFK. The proposed research will define the role of the flexible C-terminal tail in the regulation of each PFK isoform using mutagenesis and kinetic assays. Finally, PFKL has been demonstrated to oligomerize into filaments. It is hypothesized that PFKM can also oligomerize into filaments. The proposed research will identify PFKM filaments in cells, structurally charac- terize the filament, and define the role of filaments in PFKM regulation. The goal of this research is to better define mechanisms of PFK regulation. The applicant’s long-term goals involve using cryo-EM and cell-based assays as an independent researcher. This fellowship will help to prepare the applicant for a productive career as an independent researcher using cryo-EM and cell-based assays to study important biological questions.