A kinetic analysis of the folding of human carbonic anhydrase II and its catalysis by cyclophilin.
The kinetics of unfolding and refolding of human carbonic anhydrase II (HCAII) and its catalysis by the peptidyl-prolyl-cis/trans-isomerase cyclophilin were investigated. HCAII contains 15 trans- and 2 cis-prolyl peptide bonds, and, when long-term denatured, virtually all unfolded molecules contain non-native prolyl isomers. In unfolding these molecules (Us) are produced slowly in a biphasic process reflecting the isomerization of several trans-prolines and of one cis-proline. In refolding, the rapid formation of an intermediate of the molten globule type is followed by several slow prolyl isomerizations, which determine the rate of reactivation. By a short 10-s incubation in 5.0 M guanidinium chloride at 2 degrees C, unfolded HCAII species with all prolines still in the native conformation (Uf) could be produced. Surprisingly, only a fraction of Uf refolds rapidly, but the other molecules refold slowly. Evidently, some prolyl peptide bonds isomerize early in refolding, at the stage of the molten globule and as a consequence, molecules with incorrect prolyl isomers are formed in competition with the productive folding of Uf. This fraction of slow-folding molecules is strongly increased when cyclophilin is present, because it accelerates the formation of non-native prolyl isomers as long as the molecules remain in the molten globule state. Later cyclophilin catalyzes the isomerization of these prolyl peptide bonds toward the native state, which are stabilized in their conformation by further folding to the native state. This catalysis is very efficient, because only prolines that are accessible in the molten globule are involved in this sequence of isomerization and reisomerization.
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Max-Planck-Arbeitsgruppe Enzymologie der Peptidbindung, Halle/Saale, Germany.
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Schmid
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Fischer
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D000591:Amino Acid Isomerases / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D019696:Peptidylprolyl Isomerase; D011489:Protein Denaturation; D017510:Protein Folding
D002352:Carrier Proteins; D002256:Carbonic Anhydrases; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase