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 i nfrared difference spectra (RIDS). The photochemical release of ATP fr om 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 inf rared changes in the amide-I region were observed, indicating that abo ut 5-10 amino acid residues were involved in the nucleotide-binding si te. These infrared changes were due to the structural alteration of th e peptide backbone caused by the nucleotide-binding and to the couplin g 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 gr oup of Asp or Glu, as evidenced by the appearance of the 1733-cm(-1) b and, which was not observed with the AK . Mg . ADP, AK . Mg . ADP . Ar g 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) (negativ e) and at 1613 cm(-1) (positive), which were not seen in the RIDS of o ther complexes (without NO3- or/and Arg). In the transition-state-anal og complex of AK, no protonation of the carboxylate residue (Asp or Gl u) was observed, and the binding site of NO3- or the gamma-phosphate g roup of nucleotide was altered.