Kinetic analysis of cyclophilin-catalyzed prolyl cis/trans isomerization by dynamic NMR spectroscopy.
To investigate the kinetics of the prolyl peptide bond cis/trans isomerization of N-succinyl-Ala-Phe-Pro-Phe-(4)-nitroanilide catalyzed by peptidyl prolyl cis/trans isomerases (PPIases), one-dimensional dynamic 1H NMR spectroscopy was employed. To this end line shape analyses of proton signals were performed at various concentrations of both cytosolic porcine kidney cyclophilin (Cyp18) and peptide substrate. Catalysis of the cis/trans isomerization by Cyp18 is best described by a four-site exchange model, where the four sites represent the cis and trans isomers free in solution and bound to the enzyme. Combination of dynamic NMR spectroscopy with the classical protease-coupled PPIase assay allowed determination of the complete set of the microscopic rate constants describing the four site exchange model. The comparison of the rate constants of cis-->trans isomerization of the peptide free in solution and bound to cyclophilin yields an acceleration factor of 3.5 x 10(5). Dissociation of the Michaelis complexes are of the same order of magnitude as the isomerization rates on the enzyme. Therefore, all microscopic rate constants contribute to the steady state parameters. For the first time, the kcat (620 s-1) and KM (220 microM) value for the trans isomer in addition to the values of the cis isomer (kcat = 680 s-1, KM = 80 microM) could be determined under reversible conditions at pH 6.0 and 10 degrees C. The affinity of Cyp18 for the cis isomer is 4 times higher than for the trans isomer. This results in a shift of the cis/trans equilibrium toward the cis isomer.(ABSTRACT TRUNCATED AT 250 WORDS)
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Arbeitsgruppe Enzymologie der Peptidbindung, Max-Planck-Gesellschaf zur Forderung der Wissenschaften e. V., Halle/Saale, Germany.
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Scherer
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Fischer
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Drakenberg
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D000591:Amino Acid Isomerases / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D018833:Chaperonins / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D019696:Peptidylprolyl Isomerase; D011392:Proline; D013237:Stereoisomerism; D013379:Substrate Specificity; D013552:Swine
D002352:Carrier Proteins; D009842:Oligopeptides; D011392:Proline; D018833:Chaperonins; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase