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.

Kinetic properties of the monomeric enzyme dUTPase from herpes simplex virus type 1 (HSV) were investigated and compared to those previously determined for homotrimeric dUTPases of bacterial and retroviral origins. The HSV and Escherichia coli dUTPases are equally potent as catalysts towards the native substrate dUTP with a k cat /K M of about 10 7 M 31 s 31 and a K M of 0.3 W WM. However, the viral enzymes are less specific than the bacterial enzyme. The HSV and E. coli dUTPases show the same stereospecificity towards the racemic substrate analogue dUTPK KS (2P-deoxyuridine 5P-(K K-thio)triphosphate), suggesting that they have identical reaction mechanisms.z 1998 Federation of European Biochemical Societies.