Enzymes Catalyzing Peptide Bond Cis-Trans Isomerizations

Enzymes Catalyzing Peptide Bond Cis-Trans Isomerizations... 

Despite the amount of data and the simplicity of the chemical reaction catalyzed, the molecular basis of the catalytic mechanism of PPIases and APIases is still only poorly understood [155]. The considerable degree of amino acid sequence dissimilarity between the subgroups of peptide bond cis-trans isomerases also raises the challenging question of the mechanistic relatedness among the enzymes. At present there is a lack of detailed mechanistic investigations on APIases and multidomain PPIases. Thus, prototypic PPIases of all three families serve as the bases for unraveling catalytic pathways. One or more potential transition-state structures for enzyme-catalyzed prolyl isomerizations, alone or in combination, are consistent with the acceleration of the spontaneous rate of prolyl isomerization (Fig. 10.4). 

The design of this successful system started with an enzyme with a ready-made peptide binding site, with well established specificity. The cyclophilins catalyze the cis-trans isomerization of peptide bonds to proline ... 

Disulfide bond formation and the cis-trans isomerization of Pro peptide bonds are catalyzed by specific enzymes. 

At least two major slow processes occur in the folding of disulfide-containing proteins the cis-trans isomerizations of Xaa-Pro peptide bonds and the formation of the correct disulfide bonds. The latter is catalyzed by protein disulfide-isomerase (PDl). This enzyme occurs at high concentration in the endoplasmic reticulum (Hawkins et al., 1991) and there is good experimental evidence that PDl is required for the de novo folding of nascent secretory proteins (Bulleid and Freedman, 1988). Cyclophilins have recently also been localized in the ER (Hasel et al., 1991) and in other compartments of the secretory pathway (Caroni et al., 1991). Their biological function is not known. 

The first enzyme to catalyze prolyl isomerization was identified by Fischer and colleagues (1984). This discovery was possible because they developed an ingenious assay for prolyl isomerases based on the conformational specificity of chymotrypsin. This protease cleaves a chro-mogenic reporter group from a tetrapeptide (such as succinyl-Ala-Ala-Pro-Phe-4-nitroanilide) only when the Ala—Pro bond of this peptide is in the trans conformation. In aqueous solution the assay peptide exists as a 90 10 mixture of molecules with the Ala—Pro bond in trans and cis, respectively. Therefore, in the presence of a high concentration of chymotrypsin, 90% of the peptide molecules are cleaved within the dead time of manual mixing. Hydrolysis of the remaining 10% is slow because it is limited in rate by the cis —trans isomerization of the Ala—Pro bond. Acceleration of this reaction serves as a sensitive probe for prolyl isomerase activities. 

Prolyl isomerases are enzymes. In protein folding they catalyze cis trans isomerizations in both directions (Miicke and Schmid, 1992) and show equal efficiencies in unfolding and refolding experiments under identical conditions near the midpoint of the unfolding transition. They carry no information about the isomeric states of the prolyl peptide bonds in the protein substrates. The native isomer is selected by the refolding protein itself simply because the molecules... 

Common Errors - One of the most common folding errors occurs via cis-trans isomerization of the amide bond adjacent to a proline residue (see here). Proline is the only amino acid in proteins that forms peptide bonds in which the trans isomer is only slightly favored (4 to 1 versus 1000 to 1 for other residues). Thus, during folding, there is a significant chance that the wrong proline isomer will form first. It appears that cells have enzymes to catalyze the cis-trans isomerization necessary to speed correct folding. 

All X-Pro peptide bonds—where X represents any residue—are synthesized in the trans configuration. However, of the X-Pro bonds of mature proteins, approximately 6% are cis. The cis configuration is particularly common in P-turns. Isomerization from trans to cis is catalyzed by the enzyme proline-CM,tr(2 r-iso-merase (Figure 5-9). 

The prolyl isomerases catalyze isomerizations only at prolyl bonds and not at nonprolyl peptide bonds. The refolding of the P39A variant of RNase Tl, which is limited in rate by the very slow trans —> cis reisomerization of the Tyr38-Ala39 bond (see Section IV.B), is not catalyzed by cyclophilins, FKBPs, or parvulins. These enzymes are also unable to catalyze amide bond isomerizations in the proline-free model peptide Ala-Ala-Tyr-Ala-Ala (Scholz etal., 1998b). 

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