Substrate-induced fit of the ATP binding site of cytidine monophosphate kinase from Escherichia coli: Time-resolved fluorescence of 3 '-anthraniloyl-2 '-deoxy-ADP and molecular modeling

The conformation and dynamics of the ATP binding site of cytidine monophosp hate kinase from Escherichia coli (CMPKcoli), which catalyzes specifically the phosphate exchange between ATP and CMP, was studied using the fluoresce nce properties of 3'-anthraniloyl-2'-deoxy-ADP, a specific ligand of the en zyme. The spectroscopic properties of the bound fluorescent nucleotide chan ge strongly with respect to those in aqueous solution. These changes (red s hift of the absorption and excitation spectra, large increase of the excite d state lifetime) are compared to these observed in different solvents. The se data, as well as acrylamide quenching experiments, suggest that the anth raniloyl moiety is protected from the aqueous solvent upon binding to the A TP binding site, irrespective of the presence of CMP or CDP. The protein-bo und ADP analogue exhibits a restricted fast subnanosecond rotational motion , completely blocked by CMP binding. The energy-minimized models of CMPKcol i complexed with 3'-anthraniloyl-2'-deoxy-ADP using the crystal structures of the ligand-free protein and of its complex with CDP (PDB codes 1cke and 2cmk, respectively) were compared to the crystal structure of UMP/CMP kinas e from Dictyostelium discoideum complexed with substrates (PDB code 3ukd). The key residues for ATP/ADP binding to CMPKcoli were identified as R157 an d I209, their side chains sandwiching the adenine ring. Moreover, the resid ues involved in the fixation of the phosphate groups are conserved in both proteins. In the model, the accessibility of the fluorescent ring to the so lvent should be substantial if the LTD conformation remained unchanged, by contrast to the fluorescence data. These results provide the first experime ntal arguments about an ATP-mediated induced-fit of the LID in CMPKcoli mod ulated by CMP, leading to a closed conformation of the active site, protect ed from water.