High enzymatic activity and chaperone function are mechanistically relatedfeatures of the dimeric E-coli peptidyl-prolyl-isomerase FkpA

We have recently described the existence of a chaperone activity for the di meric peptidyl-prolyl cis/trans isomerase FkpA from the periplasm of Escher ichia coli that is independent of its isomerase activity. We have now inves tigated the molecular mechansim of these two activities in vitro in greater detail. The isomerase activity with a protein substrate (RNaseT1) is chara cterized by a 100-fold higher k(cat)/K-m value than with a short tetrapepti de substrate. This enhanced activity with a protein is due to an increased affinity towards the protein substrate mediated by a polypeptide-binding si te that is distinct from the active site. The chaperone activity is also me diated by interaction of folding and unfolding intermediates with a binding site that is most Likely identical to the polypeptide-binding site which e nhances catalysis. Both activities are thus mechanistically related, being based on the transient interaction with this high-affinity polypeptide-bind ing site. Only the isomerase activity, but not the chaperone activity, with the substrate citrate synthase can be inhibited by FK520. Experiments with the isolated domains of FkpA imply that both the isomerase and the chapero ne site are located on the highly conserved FKBP domain. The additional ami no-terminal domain mediates the dimerization and thus places the two active sites of the FKBP domains in juxtaposition, such that they can simultaneou sly inter act with a protein, and this is required for full catalytic activ ity. (C) 2001 Academic Press.