Peptide bond isomerization

Further examples of catalytic antibodies that are presumed to control rotational entropy are AZ-28, which catalyses an oxy-Cope [3.3]-sigmatropic rearrangement (Appendix entry 13.1) (Braisted and Schultz, 1994 Ulrich et al, 1996) and 2E4, which catalyses a peptide bond isomerization (Appendix entry 13.3) (Gibbs et al., 1992b Liotta et al., 1995). Perhaps the area for the greatest opportunity for abzymes to achieve control of rotational entropy is in the area of cationic cyclization reactions (Li et al., 1997). The achievements of the Lerner group in this area (Appendix entries 15.1-15.4) will be discussed later in this article (Section 6). 

F. X. Schmid, R. Grafl, A. Wrba, and J. J. Beintema, Role of proline peptide bond isomerization in unfolding and refolding of ribonuclease, Proc. Natl. Acad Sci. U.S.A. 83, 872-876 (1986). 

Figure 16.7 Mechanism of aspartyl proteases involving general acid-base catalysis and the formation of a protonated terahedral intermediate. Bottom Proposal by T. J. Rodriguez. T. A. Angeles, and T. D. Meek, Biochemistry 32, 12380 (1993), that the first step is peptide bond isomerization. This accounts for the observed inverse 15N/14N kinetic isotope effect, which implies that bonding with the N atom becomes stiffer in the transition state.

The importance of prolyl peptide bond isomerizations for protein folding is indicated by the following experimental observations. 

The Up Ug reactions in unfolded proteins have properties that are characteristic of prolyl peptide bond isomerizations in small peptides. The equilibrium is independent of temperature (Schmid, 1982) and independent of the concentration of additives, such as guanidinium chloride (GdmCl) (Schmid and Baldwin, 1979), that strongly decrease protein stability but do not affect prolyl peptide bond isomerization. The reaction is catalyzed by strong acid and it shows an activation energy of 88 kj/ mol, as expected for prolyl isomerization (Schmid and Baldwin, 1978). 

The refolding of the U molecules involves slow steps that are limited by prolyl peptide bond isomerization. Folding steps and prolyl isomerization steps can be mutually interdependent. On the one hand the presence of incorrect isomers in the chain can decelerate crucial folding steps, and on the other hand rapid chain folding can affect... 

The search for an enzymatic activity that would catalyze prolyl peptide bond isomerization began soon after the proposal of the proline hypothesis. The success came in 1984, when Fischer and co-workers discovered a peptidylprolyl m—tram-isomerase activity in porcine kidney and other tissues by an assay that is based on the conformational specificity of chymotrypsin. This protease cleaves the 4-nitroanilide moiety from the peptide glutaryl-Ala-Ala-Pro-Phe-4-nitroanilide only when the Ala-Pro peptide bond is in the trans conformation. In aqueous solution 90% of the molecules are trans in the assay peptide and only 10% are cis. Therefore, in the presence of a high concentration of chymotrypsin, 90% of the hydrolysis reaction occurs within the dead time of manual mixing. Hydrolysis of the remaining 10% is slow, limited in rate by the cis — ... 

Generally, multiple isomerization sites, low propensity of the thermodynamically disfavored isomer, the presence of the isomerization site in flexible protein segments, and poor dispersion of isomer-specific NMR chemical shifts illustrate the difficulties in the detection of native state peptide bond isomerization. However, methods have been developed to characterize native state prolyl isomerization in proteins that deviate much from equal partitioning of isomers [145],... 

Spontaneous peptide bond isomerization is relatively slow in respect to the NMR time-scale and, therefore, two resonance frequencies are observable. Nuclear Overhauser effect (NOE) patterns show the NMR couplings between neighboring atoms in space. That is why NOE spectra can be used to discriminate between the resonance patterns for the cis and trans conformers and to calculate the rate of exchange. 

In summary, prolyl isomerization is slow because the resonance energy of the C-N partial double bond must be overcome. The reaction is decelerated when the resonance is increased, such as in solvents that donate a hydrogen bond or a proton to the carbonyl oxygen. It is accelerated when the resonance is decreased, particularly by N protonation. The chemical and mechanistic aspects of peptide bond isomerization have been reviewed (Stein, 1993 Fischer, 2000). 

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