6 research outputs found
Multidimensional NMR identifies the conformational shift essential for catalytic competence in the 60-kDa Drosophila melanogaster dUTPase trimer
The catalytic mechanism of dUTP pyrophosphatase (dUTPase), responsible
for the prevention of uracil incorporation into DNA, involves ordering
of the flexible C terminus of the enzyme. This conformational shift is
investigated by multidimensional NMR on the Drosophila enzyme. Flexible
segments of the homotrimer give rise to sharp resonances in the
H-1-N-15 heteronuclear single-quantum coherence (HSQC) spectra, which
are clearly distinguishable from the background resonances of the well
folded protein globule. Binding of the product dUMP or the analogues
dUDP and alpha,beta-imino-dUTP to the enzyme induces a conformational
change reflected in the disappearance of eight sharp resonances. This
phenomenon is interpreted as nucleotide binding-induced ordering of
some residues upon the folded protein globule. Three-dimensional
N-15-edited H-1-N-15 HSQC total correlation spectroscopy (TOCSY) and
H-1-N-15 HSQC nuclear Overhauser effect spectroscopy measurements
allowed clear assignment of these eight specific resonance peaks. The
residues identified correspond to the conserved C-terminal sequence
motif, indicating that (i) this conformational shift is amenable to NMR
studies in solution even in the large trimeric molecule and (ii)
formation of the closed enzyme conformer in the case of the Drosophila
enzyme does not require the complete triphosphate chain of the
substrate. NMR titration of the enzyme with the nucleotide ligands as
well as kinetic data indicated significant deviation from the model of
independent active sites within the homotrimer. The results suggest
allosterism in the eukaryotic dUTPase