Structural basis for regulation and higher order assembly of human phosphofructokinase-1

Abstract

Phosphofructokinase-1 (PFK-1) is a key regulatory enzyme in the glycolytic pathway that catalyzes the ATP-dependent phosphorylation of fructose-6-phosphate (F6P) to generate fructose-1,6-bisphosphate (F16BP). The reaction PFK1 catalyzes is irreversible and marks the committed step in glycolysis. As the major regulator of glycolysis, PFK-1 is extensively regulated by several allosteric effectors to control glycolytic flux in the cell. Humans have three genes that encode the PFK1 muscle (PFKM), platelet (PFKP), and liver (PFKL) isozymes. Each isozyme has distinct roles in their respective tissues leading to different regulatory and kinetic properties. Due to a lack of structural data surrounding the human isoforms of PFK-1, the molecular mechanisms underlying these functional differences among PFK-1 isoforms have not been determined. In this work, I employed cryo-electron microscopy (cryo-EM) to study the structural basis of regulation of the PFK-1 isozymes. I solved high resolution structures of both PFKL and PFKM in functionally important liganded states. These structures show both isoforms can form filaments, and that PFKL C-terminus positioning assists in stabilizing the inactive state. Additionally, the inactive PFKL structure revealed multiple inhibitory ATP binding sites.