Conformational changes of arginine kinase induced by photochemical release of nucleotides from caged nucleotides - An infrared difference-spectroscopy investigation

The conformations of arginine kinase (AK) in AK . Mg . ADP, AK . Mg . ATP, AK . Mg . ADP . NO3- AK . Mg . ADP . Arg and AK . Mg . ADP NO3- . Arg complexes were investigated by measuring their reaction-induced infrared difference spectra (RIDS). The photochemical release of ATP from ATP[Et(PhNO(2))] and of ADP from ADP[Et(PhNO(2))] produced distinct RIDS of AK complexes, suggesting that binding of ADP and ATP promoted different structural alterations of the enzyme active-site. Small infrared changes in the amide-I region were observed, indicating that about 5-10 amino acid residues were involved in the nucleotide-binding site. These infrared changes were due to the structural alteration of the peptide backbone caused by the nucleotide-binding and to the coupling effects between the nucleotide-binding site and the other substrate (Arg or NO3-)-binding site. ATP binding to AK (as well as ADP-binding to AK in the presence of NO3-) induced protonation of a carboxylate group of Asp or Glu, as evidenced by the appearance of the 1733-cm(-1) band, which was not observed with the AK . Mg . ADP, AK . Mg . ADP . Arg and AK . Mg . ADP NO3- Arg complexes. The RIDS of the AK . Mg . ADP . NO3- . Arg complex showed new infrared bands at 1622 cm(-1) (negative) and at 1613 cm(-1) (positive), which were not seen in the RIDS of other complexes (without NO3- or/and Arg). In the transition-state-analog complex of AK, no protonation of the carboxylate residue (Asp or Glu) was observed, and the binding site of NO3- or the gamma-phosphate group of nucleotide was altered.