THE COMPLETE TRIPHOSPHATE MOIETY OF NONHYDROLYZABLE SUBSTRATE-ANALOGSIS REQUIRED FOR A CONFORMATIONAL SHIFT OF THE FLEXIBLE C-TERMINUS IN ESCHERICHIA-COLI DUTP PYROPHOSPHATASE

The molecular mechanism of substrate analogue interaction with Escheri chia coli dUTPase mas investigated, using the non-hydrolyzable 2'-deox yuridine 5'-(alpha,beta-imido)triphosphate (alpha,beta-imido-dUTP), Bi nding of this analogue induces a difference in the far UV circular dic hroism (CD) spectrum arguing for a significant change in protein confo rmation, The spectral shift is strictly Mg2+-dependent, does not appea r with dUDP instead of alpha,beta-imido-dUTP and is not elicited if th e flexible C-terminal arm is deleted from the protein by limited trypt ic digestion, Involvement of the C-terminal arm in alpha,beta-imido-dU TP binding is consistent with the finding that this analogue protects against tryptic hydrolysis at Arg-141. Near UV CD of ligand-enzyme com plexes repeals a characteristic difference in the microenvironments of enzyme-bound dUDP and alpha,beta-imido-dUTP, a difference not observa ble in C-terminally truncated dUTPase, The results suggest that (i) cl osing of the active site during the catalytic cycle, through the movem ent of the C-terminal arm, requires the presence of the complete triph osphate moiety of the substrate in complex with Mg2+,and (ii) after ca talytic cleavage the active site pops open to facilitate product relea se, (C) 1998 Federation of European Biochemical Societies.