CYCLOPHILIN ACTIVE-SITE MUTANTS HAVE NATIVE PROLYL ISOMERASE ACTIVITYWITH A PROTEIN SUBSTRATE

The prolyl isomerase activity of cyclophilins is traditionally measure d by an assay in which prolyl cis/trans isomerization in a chromogenic tetrapeptide is coupled with its isomer-specific cleavage by chymotry psin. Two variants of mitochondrial cyclophilin with substitutions in the presumed active site (R73A and H144Q) are inactive in the protease -coupled assay, but show almost wild-type activity in an assay that is based on the catalysis of a proline-limited protein folding reaction. This prolyl isomerase assay is preferable, both because coupling with proteolysis is avoided and because an intact protein instead of a sho rt peptide is used as a substrate. Possibly, some earlier conclusions about the catalytic mechanism and the involvement of the prolyl isomer ase activity in the cellular function of immunophilins may need reeval uation. (C) 1997 Federation of European Biochemical Societies.