Bradykinin and its Gly6 analogue are substrates of cyclophilin: a fluorine-19 magnetization transfer study.

Fluorine-19 magnetization transfer experiments have been used to determine the rates of cis/trans isomerization about the X-Pro7 peptide bond in [p-fluoro-Phe8]bradykinin (cis/trans ratio approximately 0.1) and its Gly6 analogue (cis/trans ratio approximately 0.4). The measurements were carried out both prior to and after the addition of cyclophilin, which has recently been shown to have peptidyl-proline cis/trans isomerase activity and is the apparent target enzyme of the immunosuppressive agent cyclosporin A. Magnetization transfer measurements over the temperature range 40-75 degrees C in the absence of enzyme give activation energies of 22.8 and 23.0 kcal/mol for [p-fluoro-Phe8]bradykinin and its Gly6 analogue, respectively. The values for the uncatalyzed cis----trans rate constant, kc, are determined by extrapolation to be 4.8 x 10(-2) and 2.1 x 10(-2) s-1 for the two peptides at 25 degrees C. The enzyme-catalyzed enhancement of the cis/trans interconversion rate was proportional to added cyclophilin concentration and was strongly sequence specific, with bradykinin a much better substrate than [Gly6]bradykinin. At a peptide concentration of 2.2 mM, the catalytic activity expressed as kc per micromolar cyclophilin was determined to be 1.2 s-1/microM for [p-fluoro-Phe8]bradykinin and 0.13 s-1/microM for the Gly6 analogue. The increased cis----trans interconversion rates were strongly inhibited by cyclosporin A and the 6-(methylalanine) derivative, which bind to cyclophilin, but not by the 1-(tetrahydrofurfuryl) derivative of cyclosporin that binds weakly.