Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy.
Purine nucleoside phosphorylase (PNP) is a key enzyme of the nucleoside salvage pathway and is characterized by complex kinetics. It was suggested that this is due to coexistence of various oligomeric forms that differ in specific activity. In this work, the molecular architecture of Escherichia coli PNP in solution was studied by analytical ultracentrifugation and CD spectroscopy. Sedimentation equilibrium analysis revealed a homohexameric molecule with molecular mass 150+/-10 kDa, regardless of the conditions investigated-protein concentration, 0.18-1.7 mg/mL; presence of up to 10 mM phosphate and up to 100 mM KCl; temperature, 4-20 degrees C. The parameters obtained from the self-associating model also describe the hexameric form. Sedimentation velocity experiments conducted for broad protein concentration range (1 microg/mL-1.3 mg/mL) with boundary (classical) and band (active enzyme) approaches gave s(0)20,w=7.7+/-0.3 and 8.3+/-0.4 S, respectively. The molecular mass of the sedimenting particle (146+/-30 kDa), calculated using the Svedberg equation, corresponds to the mass of the hexamer. Relative values of the CD signal at 220 nm and the catalytic activity of PNP as a function of GdnHCl concentration were found to be correlated. The transition from the native state to the random coil is a single-step process. The sedimentation coefficient determined at 1 M GdnHCl (at which the enzyme is still fully active) is 7.7 S, showing that also under these conditions the hexamer is the only catalytically active form. Hence, in solution similar to the crystal, E. coli PNP is a hexameric molecule and previous suggestions for coexistence of two oligomeric forms are incorrect.
Modrak-Wójcik
Anna
A
Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Poland.
Stepniak
Katarzyna
K
Akoev
Vladimir
V
Zółkiewski
Michał
M
Bzowska
Agnieszka
A
D002942:Circular Dichroism; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry; D008970:Molecular Weight; D011487:Protein Conformation; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry*; D014461:Ultracentrifugation
D029968:Escherichia coli Proteins; D011683:Purine-Nucleoside Phosphorylase