A catalytic site of protein disulfide isomerase probed with adenosine-5′-triphosphate analogs

Anthraniloyl adenosine-5'-triphosphate (Ant-ATP) and etheno-adenosine-5'-triphosphate (epsilon-ATP) complexed to Mg2+ ions are substrates of protein disulfide isomerase (PDI). epsilon-ATP, coordinated to Tb3+ ions, was used as a probe of the ATPase binding site. Sensitized luminescence arising from resonance energy transfer from epsilon-adenine to Tb3+ is quenched by PDI. The luminescence results are discussed in reference to a model in which the distance of separation between epsilon-adenine (donor) and Tb3+ (acceptor) is increased upon binding of PDI. The interaction of a small peptide of 14 amino acid residues with the b/b' domain of the protein does not influence the ATPase activity. The phosphorescence, fluorescence and fluorescence anisotropy of bound epsilon-ATP are not perturbed by the binding of the small molecular weight peptide to PDI. It is suggested that the peptide and ATP do not share a common binding site on the b/b' domain. (C) 2000 Elsevier Science B.V. All rights reserved.