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 SlyD 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.
Janowski
B
B
Department of Enzymology of Protein Folding, Max Planck Research Unit, Halle, D-06120, Germany.
Wöllner
S
S
Schutkowski
M
M
Fischer
G
G
D002384:Catalysis; D016572:Cyclosporine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000032:analysis* / Q000037:antagonists & inhibitors; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D009842:Oligopeptides; D010455:Peptides; D016572:Cyclosporine; D019696:Peptidylprolyl Isomerase