The complete triphosphate moiety of non‐hydrolyzable substrate analogues is required for a conformational shift of the flexible C‐terminus in E. coli dUTP pyrophosphatase

Abstract

The molecular mechanism of substrate analogue interaction with Escherichia coli dUTPase was investigated, using the non-hydrolyzableBinding of this analogue induces a difference in the far UV circular dichroism (CD) spectrum arguing for a significant change in protein conformation. The spectral shift is strictly Mg 2+ -dependent, does not appear with dUDP instead of K K,L L-imido-dUTP and is not elicited if the flexible C-terminal arm is deleted from the protein by limited tryptic digestion. Involvement of the C-terminal arm in K K,L Limido-dUTP binding is consistent with the finding that this analogue protects against tryptic hydrolysis at Arg-141. Near UV CD of ligand-enzyme complexes reveals a characteristic difference in the microenvironments of enzyme-bound dUDP and K K,L L-imido-dUTP, a difference not observable in C-terminally truncated dUTPase. The results suggest that (i) closing of the active site during the catalytic cycle, through the movement of the C-terminal arm, requires the presence of the complete triphosphate moiety of the substrate in complex with Mg 2+ , and (ii) after catalytic cleavage the active site pops open to facilitate product release.z 1998 Federation of European Biochemical Societies.

Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

“…This conformational change is realized by intricate subunit interactions as the C terminus of one subunit crosses over its neighboring subunit and adopts an ordered conformation to contact the substrate bound in the active site of the third subunit. In the bacterial enzyme, detailed functional investigations (2,30,33,34), in agreement with the crystal structure in complex with the nucleotide dUDP (28), indicate that the ␥ phosphate of the substrate is indispensable in inducing the closed conformer, and after catalytic cleavage the active site pops open. In the case of the human enzyme, the crystal structure suggests the retaining of the closed conformer even with the product dUMP (24), but there are no functional data published.…”

“…1 Binding Affinity of D. melanogaster dUTPase toward dUDP and dUMP-The nonhydrolyzable character of dUDP by D. melanogaster dUTPase, as established in the previous section, identifies this nucleoside diphosphate as a close substrate analogue useful for detailed analysis of the active site in equilibrium complexation experiments. In previous studies from this laboratory, CD spectroscopy was found useful for exploring the interaction of dUTPase from E. coli with dUDP (30,33,39). Following these earlier observations, spectra were recorded for the D. melanogaster dUTPase enzyme, for dUDP, and for an equimolar mixture thereof ( Fig.…”

“…Phosphate recognition is provided conserved motifs of the neighboring subunit. The third subunit contributes its C-terminal fifth conserved motif, the ordering of which upon the active site induces the catalytically competent closed conformation (24,29,30). Despite the overall similarities, the available data suggest the existence of marked differences between prokaryotic and eukaryotic dUTPases.…”

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

“…The measured binding affinities and catalytic rates of the T138Stop and H145A enzymes are consistent with the essential role of the C-terminal arm in other dUTPase proteins investigated. 17,25,[27][28][29][30][31][32] To better understand the underlying energetic and structural causes determining the catalytic activity, we carried out QM/MM calculations of the reaction mechanism for proton transfer and phosphate cleavage in the WT and the two mutant (H145A and T138Stop) enzyme complexes. We recently identified a one-step associative A N D N catalytic mechanism for the hydrolysis at the α-phosphate, involving also a coupled proton transfer step from the nucleophilic water to the catalytic Asp83 residue.…”

“…Interestingly, the particularly important role of the A2 and A3 angles involving the γ-phosphate ( Figure 8B) could contribute to the dUDP/dUTP specificity via the C-terminal arm, preventing the dUDP hydrolysis while allowing it for dUTP. For E. coli enzymes, it was found that the flexible C-terminal arm takes up an ordered structure only in the presence of the γ-phosphate, [29][30] whereas other organisms such as the human dUTPase may have a C-terminal arm located in the vicinity of the active site, even when a bound substrate is absent. 69 A recent mutational study demonstrated that the C-terminal arm P-loop-like motif is required for catalytic activity and it also helps to discriminate against dUDP substrates.…”