Structural and functional characterization of Escherichia coli peptidyl-prolyl cis-trans isomerases.
Peptidyl-prolyl cis-trans isomerases (PPIases), enzymes that catalyze the cis-trans isomerization of peptide bonds to which proline contributes the nitrogen, were purified from Escherichia coli. In this organism, at least two PPIases are present. Both the cationic (periplasmic) and anionic (cytoplasmic) PPIases are inhibited by cyclosporin A with a Ki of 25-50 microM, a concentration 1000-fold higher than that required for eukaryotic PPIases. Although isoelectric focusing indicates that the two enzymes differ in isoelectric point by at least 4.0 pH units, the specific activities of the enzymes toward the tetrapeptide substrate succinyl-Ala-Ala-Pro-Phe-methyl-coumarylamide are equivalent. The activity of both enzymes for a series of substituted succinyl-Ala-Xaa-Pro-Phe-para-nitroanilide tetrapeptides suggests that the structure and function of the active site of the prokaryotic proteins is similar to that of eukaryotic cyclophilins. Both enzymes are capable of catalyzing the refolding of thermally denatured type III collagen. Antibodies against the periplasmic PPIase do not recognize the cytoplasmic enzyme, indicating significant differences in epitopes between the two forms. Circular dichroism spectroscopy indicates that the secondary structure of the cationic protein consists of 17% alpha-helix, 34% beta-sheet, 17% turns, 33% random coil and is very similar to human cytosolic PPIase.