A protease-free assay for peptidyl prolyl cis/trans isomerases using standard peptide substrates

Peptidyl prolyl cis/trans isomerases (PPIases) are ubiquitous and abundant enzymes catalyzing peptide bond cis/trans isomerization adjacent to proline in peptides and proteins. An uncoupled protease-free assay of PPIase activity has been developed using the standard tetrapeptide substrates of the proteolytically coupled test system. Differences in the UV/vis absorption spectra of cis and trans conformations of Suc-Ala-Xaa-Pro-Phe-(Y-) anilide (Xaa = Ala, Leu, Phe; Y = 4-nitro, 2,4-difluoro) were exploited to monitor the time course of the cis/trans isomerization subsequent to a solvent jump from 0.47 M LiCl/trifluoroethanol into aqueous solution. The utility of the assay has been demonstrated by the determination of the Michaelis-Menten constants of cytosolic cyclophilin (Cyp18) and of the proteolytically sensitive FK506-binding protein-like PPIase Sly D from Escherichia coli. Furthermore, similar inhibition constants were estimated for the reversible inhibition of human Cyp18 by cyclosporin A (CsA) with both the proteolytically coupled and the novel uncoupled PPIase assay. (C) 1997 Academic Press.