Prolyl isomerases peptide bond isomerization

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 — ... 

Clearly, the action of prolyl isomerases is not restricted to the slow folding of polypeptide chains with intact disulfides, but they also accelerate the oxidative folding of reduced proteins, which resemble more closely the nascent polypeptide chains as they occur in the endoplasmic reticulum. The simultaneous presence of PPI markedly enhances the efficiency of PDI as a catalyst of disulfide bond formation. Both enzymes act according to their specificity and catalyze the isomerization of prolyl peptide bonds and the formation of disulfide bonds, respectively, in the folding protein chains. It remains to be demonstrated that a similar concerted action of the two enzymes can take place in the course of de novo synthesis and folding of proteins in the cell. 

The peptidyl-prolyl cis/trans isomerase Pin 1 isomerizes the peptide bond of a phos-phorylated serine or phosphorylated threonine followed by a proline. Through isomerization of pSer-Pro or PThr-Pro, Pinl regulates a number of proteins. Together with its ability to regulate phosphorylation and conformation of tau proteins. Pin 1 is considered a potential neuroprotective function against AD. 

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]. 

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). 

A variant of the peptide-based assay avoids the coupling with isomer-specific proteolysis (Janowski et al., 1997). The absorbance at 330 nm of succinyl-Ala-Phe-Pro-Phe-4-nitroanilide decreases slightly when the Phe and the nitroanilide moieties rearrange on the cis —trans isomerization at the Phe-Pro bond of the peptide. Because of the small change in signal, the sensitivity of the assay is limited, but it is the method of choice for assaying prolyl isomerases sensitive to proteases. 

Recently, Kiillertz and Fischer developed a new prolyl isomerase assay for high-troughput screening of up to 20,000 samples per day. This assay employs a disulfide-bonded cis peptide with internally quenched fluorescence. Cis/trans isomerization is initiated by rapid reductive ring opening and followed by the increase in fluorescence (G. Kiillertz and G. Fischer, unpublished). 

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... 

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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