Functional Analysis of Conformational Changes of Phosphofructokinase

Abstract

The glycolytic flux is tightly regulated in unicellular and multicellular organisms under anaerobic conditions. A main regulatory step is centered around the behavior of phosphofructokinase (PFK), which catalyzes the phosphorylation of fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate in the presence of adenosine triphosphate (ATP). PFK shows a species dependent subunit composition, oligomerization state, and specific kinetic behavior. Monomer sizes range from 34kDa in prokaryotes to 110kDa in eukaryotes. The diversity of composition and oligomerization state allows for subtle differences in the catalytic and regulatory mechanism of this enzyme in different organisms. The most knowledge has been obtained for the bacterial PFKs, which are homotetramers with a molecular mass of 140 kDa. A combination of X-ray crystallographic and biochemical data has provided an understanding of the conformational changes that occur between the active and inactive states and the role of the only two bacterial effectors [1]. Inactivation is achieved by a 7°rotation of the subunits in the plane perpendicular to the catalytic surface. Eukaryotic PFKs have a higher level of regulatory complexity facilitated by an evolutionary process of gene duplication and fusion of the bacterial gene that has given rise to a "double-size" eukaryotic enzyme.Among all the eukaryotic PFKs, the one from S. cerevisiae (ScPFK) is the best characterized. This enzyme is a hetero-octamer of 835 kDa, composed of 4α-and 4β-subunits of approximately 105 kDa each. Like its prokaryotic counterpart, it shows cooperativity for F6P and non-cooperativity for ATP. Most insights into the structural/functional mechanism of these enzymes have been obtained by 3DEM. The 4α-subunits of the ScPFK form the inner core of the enzyme onto which the 4β-subunits are bound [2]. These 3DEM studies permitted to correctly infer the composition and the localization of the catalytic binding region [3] and combined with molecular replacement aided the determination the X-ray structure of the ScPFK truncated tetramer in the presence of F6P [4]. The cryo 3DEM structure of ScPFK in the inactive state [5], in combination with studies of other yeast structures in different states, has manifested a strong correlation between structural conformation and kinetic behavior. The structural studies have shown that minor subunit rearrangements influence the rotation between the tetramers in the octameric complex, correlated with the kinetic behavior of the enzyme. However, there is still a lack of understanding of the small structural changes that lead to this large conformational change.We have analyzed the ScPFK in the presence of either F6P or ATP to understand the structural changes taking place when the enzyme switches from the active to the inactive state. Cryo 3DEM data were collected for the enzyme in both states (Fig. 1, Top) and 3D reconstructions, with 1.3 nm resolution, were calculated by 3D reference based projection alignment methods using Radon transform algorithms (Fig. 1, Cent...