Ordering of the peptide-like C-terminal tail of dUTPase upon binding of substrate analogues:a heteronuclear NMR study

摘要

Homotrimeric dUTP pyrophosphatase is an indispensible enzyme involved in the regulation of dUTP/dTTP ratio in the cell,thus playing a key role of preventing 'thymine-less cell death'.The Drosophila enzyme,due to its size(3x187 amino acid residues)is not suitable for conventional(i.e.non-TROSY)NMR at 500 MHz.Howver,the C-terminal part of the subunits is highly disordered in the apoenzyme,giving rise to sharp resonances.Upon titration with the substrate analogues dUMP,dUDP and alpha,beta-imino dUTP a speficic set of peaks 'disappears' from the HSQC spectrum indicating that the corresponding region becomes ordered in the course of catalysis.Further heteronuclear techniques allowed more detailed characterization of the changes upon substrate/analogue binding.