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‐hydrolyzable 2′‐deoxyuridine 5′‐(α,β‐imido)triphosphate (α,β‐imido‐dUTP). Binding 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 α,β‐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 α,β‐imido‐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 α,β‐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.