MECHANISTIC STUDIES OF ENZYMATIC AND NONENZYMIC PROLYL CIS-TRANS ISOMERIZATION

The cyclosporin A binding protein, cyclophilin (CyP), and the FK-506 binding protein, FKBP, catalyze the cis-to-trans isomerization of Xaa-Pro bonds in peptides. To probe the mechanism of these reactions and their nonenzymatic counterparts, we determined the following: (1) substrate specificities of CyP and FKBP; (2) dependencies of k(c)/K(m) on pH and solvent deuterium; (3) secondary deuterium isotope effects; and (4) temperature-dependencies. The results indicate that (1) for cis-to-trans isomerization of Suc-Ala-Xaa-cis-Pro-Phe-pNA, values of k(c)/K(m) for the CyP-catalyzed reactions show little dependence on Xaa. In contrast, for FKBP, k(c)/K(m) displays a marked dependence on Xaa with a preference for hydrophobic residues. (2) For both enzymes, k(c)/K(m) is independent of both pH and isotopic composition of the solvent. (3) For the CyP-catalyzed cis-to-trans isomerization of Suc-Ala-Gly(L,L)-cis-Pro-Phe-pNA (L = H, D), H(k(c)/K(m))/D(k(c)/K(m)) = 1.13 +/- 0.01 and is independent of temperature between 2 and 30-degrees-C (2-degrees-C, (H)k/(D)k = 1.14 +/- 0.02; 10-degrees-C, (H)k/(D)k = 1.13 +/- 0.01; 30-degrees-C, (H)k/(D)k = 1.14 +/- 0.03). A secondary deuterium isotope effect of 1.13 suggests that, in the transition state of this reaction, the Xaa-Pro bond is twisted out of planarity. This value is inconsistent with mechanisms involving nucleophilic catalysis. (4) Eyring plots of In [(k(c)/K(m))/T] vs 1/T for reactions of FKBP are linear, while Eyring plots for CyP catalysis are curved and display a maximum around 30-degrees-C. The data for CyP were fit to a model involving a temperature-dependent, reversible isomerization of active to an inactive enzyme. This model is supported by the temperature-independence of the secondary deuterium isotope effect (see 3 above) and the independence of the equilibrium of the two enzyme forms on substrate structure. The activation parameters that were calculated from the Eyring plots indicated enthalpy-entropy compensation for both CyP and FKBP with the critical temperature, T(c), equal to 287 and 260 K, respectively. We interpret the compensation in terms of a simple mechanism in which stronger transition-state interactions between enzyme and substrate (more positive DELTA-H double dagger) are accompanied by greater restrictions of translational and rotational freedom (more negative DELTA-S double dagger).