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

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• 作者：Inè ; s M. Li de la Sierra ; Jacques Gallay ; Michel Vincent ; Thomas Bertrand ; Pierre Briozzo ; Octavian Bâ ; rzu ; and Anne-Marie Gilles
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：December 26, 2000
• 年：2000
• 卷：39
• 期：51
• 页码：15870 - 15878
• 全文大小：355K
• 年卷期：v.39,no.51(December 26, 2000)
• ISSN：1520-4995

文摘

The conformation and dynamics of the ATP binding site of cytidine monophosphate kinasefrom Escherichia coli (CMPK_coli), which catalyzes specifically the phosphate exchange between ATPand CMP, was studied using the fluorescence properties of 3'-anthraniloyl-2'-deoxy-ADP, a specific ligandof the enzyme. The spectroscopic properties of the bound fluorescent nucleotide change strongly withrespect to those in aqueous solution. These changes (red shift of the absorption and excitation spectra,large increase of the excited state lifetime) are compared to those observed in different solvents. Thesedata, as well as acrylamide quenching experiments, suggest that the anthraniloyl moiety is protected fromthe aqueous solvent upon binding to the ATP binding site, irrespective of the presence of CMP or CDP.The protein-bound ADP analogue exhibits a restricted fast subnanosecond rotational motion, completelyblocked by CMP binding. The energy-minimized models of CMPK_coli complexed with 3'-anthraniloyl-2'-deoxy-ADP using the crystal structures of the ligand-free protein and of its complex with CDP (PDBcodes 1cke and 2cmk, respectively) were compared to the crystal structure of UMP/CMP kinase fromDictyostelium discoideum complexed with substrates (PDB code 3ukd). The key residues for ATP/ADPbinding to CMPK_coli were identified as R157 and I209, their side chains sandwiching the adenine ring.Moreover, the residues involved in the fixation of the phosphate groups are conserved in both proteins.In the model, the accessibility of the fluorescent ring to the solvent should be substantial if the LIDconformation remained unchanged, by contrast to the fluorescence data. These results provide the firstexperimental arguments about an ATP-mediated induced-fit of the LID in CMPK_coli modulated by CMP,leading to a closed conformation of the active site, protected from water.