High enzymatic activity and chaperone function are mechanistically related features of the dimeric E. coli peptidyl-prolyl-isomerase FkpA.
We have recently described the existence of a chaperone activity for the dimeric peptidyl-prolyl cis/trans isomerase FkpA from the periplasm of Escherichia coli that is independent of its isomerase activity. We have now investigated the molecular mechanism of these two activities in vitro in greater detail. The isomerase activity with a protein substrate (RNaseT1) is characterized by a 100-fold higher k(cat)/K(M) value than with a short tetrapeptide substrate. This enhanced activity with a protein is due to an increased affinity towards the protein substrate mediated by a polypeptide-binding site that is distinct from the active site. The chaperone activity is also mediated by interaction of folding and unfolding intermediates with a binding site that is most likely identical to the polypeptide-binding site which enhances catalysis. Both activities are thus mechanistically related, being based on the transient interaction with this high-affinity polypeptide-binding 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 chaperone site are located on the highly conserved FKBP domain. The additional amino-terminal domain mediates the dimerization and thus places the two active sites of the FKBP domains in juxtaposition, such that they can simultaneously interact with a protein, and this is required for full catalytic activity.
Ramm
K
K
Biochemisches Institut, Universität Zürich, Winterthurerstr. 190, Zürich, CH-8057, Switzerland.
Plückthun
A
A
D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000378:metabolism; D017124:Conserved Sequence; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006193:Haemophilus influenzae / Q000201:enzymology; D020104:Immunophilins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D006163:Ribonuclease T1 / Q000737:chemistry / Q000378:metabolism; D016559:Tacrolimus / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; C058028:immunomycin; D002950:Citrate (si)-Synthase; D006163:Ribonuclease T1; D022061:Tacrolimus Binding Protein 1A; D020104:Immunophilins; D019696:Peptidylprolyl Isomerase; C068254:FkpA protein, E coli; D016559:Tacrolimus