Secondary Amide Peptide Bonds

Imidic and Secondary Amide Peptide Bond Conformation... 

Despite this low thermodynamic stability the permanent existence of a single secondary amide peptide bond in cis conformation per 1000 amino acid residues is the minimal population that has to be considered for unfolded polypeptide chains. This cis peptide bond fluctuates across the polypeptide chain in relation to the sequence-specific propensity of a secondary amide peptide bond to adopt the cis conformation. As could be found in folded proteins, nonprolyl cis peptides are frequently located in the fS-region of a q>/y/ plot [22]. It was hypothesized that cis peptide bonds represent high-energy structures able to store potential energy for increasing chemical reactivity [23]. Interconversion rates for the reversible CTI of secondary amide peptide bonds typically lead to half times of about 1 s for dipeptides, which decreases about 4-fold when the peptide bond is positioned in the middle of a longer peptide chain. 

Despite the similarity of cis to trans interconversion rates, secondary amide peptide bonds and imidic peptide bonds differ greatly in their capacity to form the cis isomeric state. This means that the decreased cis population of secondary amide peptide bonds results from higher rates for CTIs. In the case of the Ala-Tyr dipeptide a 250-fold difference for the interconversion rate constants was observed (kc t = 0.6 s-1, kt c = 2.4 x 10-3 s-1, 298 K) [21]. Generally, chain elongation causes destabilization of the cis conformation when located apart from charged termini, and thus leads to a decreased cis population as well as a lowered barrier to rotation in cis to trans direction. 

Interaction of peptides with Li+ ions in dry solvents, such as trifluorethanol (TFE) and tetrahydrofurane, can dramatically influence the free energy difference that discriminates the cis from the trans isomers for both prolyl bonds and secondary amide peptide bonds [56-59]. However, the Li+-induced increase of the cis isomer population in linear oligopeptides depends on the nature of the amino acids preceding proline (G. Fischer, unpublished results). 

By use of site-directed mutagenesis in positions covering cis prolyl bonds, the proline has been replaced by nonproline amino acids. It came as a surprise that the secondary amide peptide bond formed in the substitution still adopts the thermodynamically disfavored cis conformation in many cases [25,26,132-135], Thus, to overcome the free energy costs of a cis secondary amide peptide bond of about 15 kj mol-1 the structural consequences favoring the trans conformation must be absent in the folded protein variant. Consequently, the CTI is largely retained in these protein variants [133],... 

There is evidence that the trigger factor and the hsp70 chaperone DnaK, a PPIase and a secondary amide peptide bond cis-trans isomerase (APIase) respectively, contribute to the formation of native proteins by apparently overlapping functions with the trigger factor as the primary interaction partner of the emerging polypeptide chain [122-124]. Consequently, synthetic lethality was observed... 

Secondary Amide Peptide Bond Cis-Trans Isomerases I 213... 

The dynamics of CTI demonstrate the fundamental similarity between prolyl bonds and secondary amide peptide bonds, making it probable that enzymes exist for the rate acceleration of both types of reactions. The relatively low spontaneous rates indicate the potential importance of CTI of secondary amide peptide bonds as rate-limiting step in protein backbone rearrangements preceding the formation of biologically active proteins. 

Whereas peptidyl prolyl cis-trans isomerases constitute a well-characterized enzyme class comprising well over 1000 members with small sequence variations in the proteins of different species, the discovery of secondary amide peptide bond cis-trans isomerases (APIases) had to await the development of suitable enzyme assays. Fortunately, spectral differences in the UV region between cis and trans isomers of dipeptides could be exploited to identify and quantify isomerization rate-enhancing factors in biological material [149]. 

Recombinantly produced DnaK was utilized to characterize its APIase function enzymatically. Co-chaperones, such as DnaJ and GrpE in the presence of ATP might contribute to create the subsite specificity of DnaK. For oligopeptide substrates, the APIase function of DnaK does not require concomitant ATP hydrolysis. A functional overlap of a PPIase and an APIase, trigger factor and DnaK, respectively, could not be observed in an APIase and a standard PPIase assay [127]. Generally, PPIases fail to accelerate CTI of secondary amide peptide bonds in peptide substrates and folding intermediates [152]. 

The design of selective and potent inhibitors of PPIases is of interest and numerous molecules have been designed or selected from chemical libraries with a view to curing these major diseases. The study of Pinl, which is clearly distinct from other members of the PPIase family on the basis of structure, binding site, catalytic mechanism, and biological implications, has opened up new perspectives in the biological chemistry of PPIases. The recent discoveries of the secondary amide peptide bond cis-trans isomerase (APIase) DnaK [209] and of a novel class of FK506 and cyclosporine-sensitive PPIase [210] are also major advances in this field. 

Beyond protein folding, the discovery of peptidyl prolyl isomerases (PPIases) and related proteins has opened the way to novel concepts in biology the notion of chaperone-assisted receptor binding is an emerging field of research which sheds light on receptor function and protein-protein interactions. The recent discovery of a secondary amide peptide bond cis-trans isomerase (APIase) heralds new advances in this field. 

---