Prolyl Isomerization

The smallest member of a new family of prolyl iso-merases (unrelated to the cyclophilins or the FK-506 binding proteins) that catalyzes the proline-limited folding of a variant of ribonuclease T1 with a KJK value of 30,000 M s With the tetrapeptide succinyl-Ala-Leu-Pro-Phe-4-nitroanilide as a substrate in parvulin-catalyzed prolyl isomerization, this parameter is 1.1 x 10 M s Parvulin also accelerates its own refolding in an autocatalytic fashion. 

Chyraotrypsin inhibitor 2 (CI2) folds rapidly by simple two-state kinetics that is, D N, with a r1/2of 13 ms.18,19 CI2 is a small 64-residue protein that has all its peptidyl-proline bonds in the favorable trans conformation.20 (There are, of course, additional slow cis —> trans peptidyl-prolyl isomerization events, which account for about 20-30% of the refolding amplitudes.) The occurrence of two-state kinetics does not prove that there are no intermediates on the folding pathway there could be intermediates that are present at high energy and are kineti-cally undetectable (see section B4). Two-state behavior has subsequently been found for many other small proteins. The simplicity of two-state folding kinetics provides the ideal starting point for the analysis and illumination of the basic principles of folding. 

Ryo, A., Suizu, F., Yoshida, Y., Perrem, K., Liou, Y.C., Wulf, G., Rottapel, R., et al. Regulation of NF-kappaB signaling by Pin 1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA. Mol Cell 12 (2003) 1413-1426. 

IV. Mechanism of Enzymatic Prolyl Isomerization Catalysis by Distortion. 21... 

In this review, I will describe our current understanding of the mechanism of both enzymatic and nonenzymatic prolyl isomerization. I will first review nonenzymatic prolyl isomerization and rotation about amide... 

To supplement the data on prolyl isomerization, I will draw on the literature describing rotation about the C-N bond in secondary amides. Early studies in this field were described by Stewart and Siddall in an excellent 1970 review. As we will see, these reactions are related to prolyl isomerization and support the mechanism to be proposed for prolyl isomerization. The mechanism is based on results from a variety of experimental approaches. In all cases, experiments employing kinetic-based probes will be used to obtain an accurate picture of the activated complex in the rate-limiting transition state. The experiments that will be described include thermodynamics, in which activation parameters (i.e., AG, AHt, and ASt) will be described solvent effects, in which the influence of organic solvents and deuterium oxide will be reviewed acid-base catalysis substituent effects and secondary deuterium isotope effects. 

Two types of solvent effects have been determined for prolyl isomerization and amide rotation (1) the effect of solvent deuterium on reaction rate and (2) the effect of organic solvents on reaction rate. Solvent deuterium isotope effects are useful tools in probing the role of proton transfer... 

Fig. 1. Enthalpy-entropy compensation for nonenzymatic prolyl isomerization (see Table I for literature references). 9, Proline-containing oligopeptides A, dipeptide , Gly-Gly-Lys-Phe-Pro. 1, Suc-Ala-Leu-Pro-Phe-pNA 2, Suc-Ala-Ala-Pro-Phe-pNA 3, Gly-Gly-Pro-Ala 4,N,N-dimethylacetamide 5,Suc-Ala-Trp-Pro-Phe-pNA 6, Suc-Ala-Gly-Pro-Phe-pNA 7, Gly-Pro 8, Ala-Pro 9, Val-Pro 10, His-Pro 11, Gly-Gly-Lys-Phe-Pro. Linear regression analysis of the data for compounds 1—6 yields a slope or critical temperature, T, of 232 29 K analysis of the data for compounds 7-11 yields = 212 27 K.

The effect of organic solvents on the rate constant for amide rotation in Af,A -dimethylacetamide (DMA) has also been investigated (Drakenberg et ai, 1972). As the solvent is changed from water to acetone to cyclohexane, first-order rate constants for rotation increase from 0.025 to 0.33 to 1.5 sec . This observation that nonpolar solvents increase reaction rates indicates that the transition state for amide rotation is nonpolar relative to the reactant state and, thus, is stabilized in nonpolar solvents. This transition state is presumably characterized by partial rotation about the amide bond. In this transition state, polar resonance structures for the amide bond no longer exist and, thus, the transition state is less polar than the reactant state. The 60-fold rate acceleration that accompanies transfer of DMA from water to cyclohexane will provide an important clue in understanding enzymatic prolyl isomerization (see below). 

Scheme 11. Mechanism for acid-catalyzed prolyl isomerization.

The single most revealing mechanistic parameter for prolyl isomerization and amide rotation is the secondary deuterium isotope effect. In general for such studies, the hydrogens on the carbon that is bonded to the carbonyl carbon of the amide or imide (the /3-hydrogens ) are substituted with deuterium and reaction rate constants are measured for... 

Secondary deuterium isotope effects have been measured for the cis-to-trans prolyl isomerization of Suc-Ala-Gly-cis-Pro-Phe-pNA (where pNA is p-nitroanilide) and C-N rotation in DMA. In the former case, the isotope effect for the two hydrogens of glycine is 1.05 0.02 (Fischer et al., 1989a Harrison et ai, 1990 Harrison and Stein, 1990a), and for C—N rotation in DMA, the isotope effect for the three hydrogens of the acetyl moiety is 1.10 0.05. Significantly, the effect for two deuteriums in DMA can be calculated to be 1.05 (Fujihara and Schowen, 1985). 

Scheme IV. Transition state structure for prolyl isomerization.

The available data suggest that in aqueous solution and at neutral pH prolyl isomerization proceeds according to a simple, one-step mechanism. Solvent water does not participate in the reaction and there is no accumulation of intermediates. The energy barrier to isomerization is enthalpic and represents the energy of resonance stabilization that is possessed by the C—N imide bond. 

In this section, we review the available data that address questions of the enzyme mechanism for prolyl isomerization. As we will see, these data overwhelmingly support a mechanism involving catalysis by distortion. 

Rate Comtants for Enzymatic and Uncatalyzed Prolyl Isomerization in Peptides of General Structure Suc-Ala-Ala-Xaa-Phe-pNA... 

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