Abstract

Glycolysis and respiration are the main pathways for energy production in living cells. Although respiration is mostly favored by multicellular organisms, muscle and cancer cells behave more like unicellular organisms using glycolysis as their main energy pathway. Deficiencies and dysfunction of glycolytic enzymes, in particular phosphofructokinase (Pfk-1), results in severe clinical syndromes and diseases (e.g., Hemolytic anemia, Tauri's disease, non-insulin dependent diabetes mellitus). Pfk-1 plays a key role in the regulation of the glycolytic pathway and its activity is controlled by a large number of allosteric effectors (~20 in eukaryotes vs 2 in bacteria). The reaction catalyzed by this enzyme represents the first irreversible step specific for glycolysis. During the past twenty years, large efforts have been devoted to comprehend the mechanisms of catalysis and regulation of phosphofructokinase. Even though the information about the bacterial enzyme represents a great advancement in understanding this step of the glycolytic pathway, our knowledge of this enzyme is still quite limited for higher organisms. Eukaryotic Pfk-1's not only differ in size and oligomerization state, but they also exhibit a concentration dependent association-dissociation behavior and a far more complex regulatory mechanism. Moreover, the structures of the eukaryotic enzymes are still unknown, in most cases due to the lack of good quality crystals for x-ray analysis.
The aim of this research is to analyze the structure of Pfk-1 from eukaryotic organisms (S. cerevisiae, S. pombe) in the presence of different combinations of effectors and substrates by novel techniques of cryo-electron microscopy of single particles and image processing, and by fitting x-ray models to the electron microscopy structures. These studies will provide significant new information regarding the structure/function relationship of the mechanism of catalysis and regulation of phosphofructokinase in eukaryotic organisms.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM069551-05S1
Application #
7924304
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Flicker, Paula F
Project Start
2009-09-30
Project End
2010-07-31
Budget Start
2009-09-30
Budget End
2010-07-31
Support Year
5
Fiscal Year
2009
Total Cost
$196,423
Indirect Cost

  Institution

Name
University of Vermont & St Agric College
Department
Physiology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405

  Publications

Yu, Lingbo; Snapp, Robert R; Ruiz, Teresa et al. (2013) Projection-based volume alignment. J Struct Biol 182:93-105
Azari, Fereshteh; Radermacher, Michael; Mintz, Keith P et al. (2012) Correlation of the amino-acid sequence and the 3D structure of the functional domain of EmaA from Aggregatibacter actinomycetemcomitans. J Struct Biol 177:439-46
Banaszak, Katarzyna; Mechin, Ingrid; Obmolova, Galina et al. (2011) The crystal structures of eukaryotic phosphofructokinases from baker's yeast and rabbit skeletal muscle. J Mol Biol 407:284-97
Yu, Lingbo; Snapp, Robert R; Ruiz, Teresa et al. (2010) Probabilistic principal component analysis with expectation maximization (PPCA-EM) facilitates volume classification and estimates the missing data. J Struct Biol 171:18-30
Clason, T; Ruiz, T; Schägger, H et al. (2010) The structure of eukaryotic and prokaryotic complex I. J Struct Biol 169:81-8
Radermacher, Michael (2009) Chapter 1 Visualizing functional flexibility by three-dimensional electron microscopy reconstructing complex I of the mitochondrial respiratory chain. Methods Enzymol 456:3-27
Yu, Chunxiao; Mintz, Keith P; Ruiz, Teresa (2009) Investigation of the three-dimensional architecture of the collagen adhesin EmaA of Aggregatibacter actinomycetemcomitans by electron tomography. J Bacteriol 191:6253-61
Benjamin, Shaun; Radermacher, Michael; Kirchberger, Jürgen et al. (2009) 3D structure of phosphofructokinase from Pichia pastoris: Localization of the novel gamma-subunits. J Struct Biol 168:345-51
Benjamin, Shaun; Radermacher, Michael; Bar, Jorg et al. (2007) Structures of S. pombe phosphofructokinase in the F6P-bound and ATP-bound states. J Struct Biol 159:498-506
Clason, Todd; Zickermann, Volker; Ruiz, Teresa et al. (2007) Direct localization of the 51 and 24 kDa subunits of mitochondrial complex I by three-dimensional difference imaging. J Struct Biol 159:433-42

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