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

Publisher Website
Google Scholar
Abstract
Fluoride-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 .apprx. 0.1) and its Gly6 analogue (cis/trans ratio .apprx. 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.degree. 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 value for the uncatalyzed cis .fwdarw. trans rate constant, kc, are determined by extrapolation to be 4.8 .times. 10-2 and 2.1 .times. 10-2 s-1 for the two peptides at 25.degree. 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/.mu.M for [p-fluoro-Phe8]bradykinin and 0.13 s-1/.mu.M for the Gly6 analogue. The increased cis .fwdarw. 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.