Prolyl Peptide Bonds

Other interesting examples of proteases that exhibit promiscuous behavior are proline dipeptidase from Alteromonas sp. JD6.5, whose original activity is to cleave a dipeptide bond with a prolyl residue at the carboxy terminus [121, 122] and aminopeptidase P (AMPP) from E. coli, which is a prohne-specific peptidase that catalyzes the hydrolysis of N-terminal peptide bonds containing a proline residue [123, 124]. Both enzymes exhibit phosphotriesterase activity. This means that they are capable of catalyzing the reaction that does not exist in nature. It is of particular importance, since they can hydrolyze unnatural substrates - triesters of phosphoric acid and diesters of phosphonic acids - such as organophosphorus pesticides or organophosphoms warfare agents (Scheme 5.25) [125]. 

Figure 5-9. Isomerization of the N-a, prolyl peptide bond from a cis to a trans configuration relative to the backbone of the polypeptide.

F. Marcus, Preferential Cleavage at Aspartyl-Prolyl Peptide Bonds in Dilute Acid , Int. J. Pept. Protein Res. 1985, 25, 542-546. 

In native collagen, all Gly-Pro and Xaa-Hyp peptide bonds are in the trans conformation, whereas in the unfolded state, a significant fraction of cis isomers populates at each Gly-Pro and Xaa-Hyp peptide bond, cis-to-trans isomerization reactions at prolyl peptide bonds are the origin for the observed slow kinetics of triple helix formation" as shown by their high activation energy ( 72 kj moG )" and their acceleration by prolyl... 

This enzyme [EC 3.4.11.9] (also known as Xaa-Pro aminopeptidase, X-Pro aminopeptidase, proline amino-peptidase, and aminoacylproline aminopeptidase) catalyzes the hydrolysis of a peptide bond at the iV-terminus of a peptide provided that the iV-terminal amino acyl residue is linked to a prolyl residue by that peptide bond. The enzyme will also act on dipeptides and tripeptides with that same restriction. Either manganese or cobalt is needed as a cofactor. This enzyme appears to be a membrane-bound system in both mammalian and bacterial cells. The protein belongs to the peptidase family M24B. 

This enzyme [EC 3.4.14.1], also called cathepsin C and cathepsin J, catalyzes the hydrolysis of a peptide bond resulting in the release of an N-terminal dipeptide, XaaXbb-Xcc, except when Xaa is an arginyl or a lysyl residue, or Xbb or Xcc is a prolyl residue. This enzyme, a member of the peptidase family Cl, is a CF-dependent lysosomal cysteine-type peptidase. 

Glutamyl endopeptidase 11 [EC 3.4.21.82], also known as glutamic acid-specific protease, catalyzes the hydrolysis of peptide bonds, exhibiting a preference for Glu-Xaa bonds much more than for Asp-Xaa bonds. The enzyme has a preference for prolyl or leucyl residues at P2 and phenylalanyl at P3. Hydrolysis of Glu-Pro and Asp-Pro bonds is slow. This endopeptidase is a member of the peptidase family S2A. 

This zinc-dependent enzyme [EC 3.4.11.1], also referred to as cytosol aminopeptidase, leucyl aminopeptidase, and peptidase S, catalyzes the hydrolysis of a terminal peptide bond such that there is a release of an N-terminal amino acid, Xaa-Xbb-, in which Xaa is preferably a leucyl residue, but may be other aminoacyl residues including prolyl (although not arginyl or lysyl). Xbb may be prolyl. In addition, amino acid amides and methyl esters are also readily hydrolyzed, but the rates with arylamides are exceedingly slow. The enzyme is activated by heavy metal ions. 

This endopeptidase [EC 3.4.24.16] catalyzes the hydrolysis of peptide bonds, preferentially cleaving neurotensin between Pro-10 and Tyr-11. However, there is no absolute requirement for a prolyl bond. 

C. Dugave, Study of the cis-trans isomerization of the amino-acyl prolyl peptide bond. Application to the design of novel inhibitors of immunophilins, Curr. Org. Chem. 6 (2002) 1397-1431. 

Refolding is generally found to proceed by a series of exponential phases. Many of these exponentials are a consequence of cis-trans isomerization about peptidyl-prolyl bonds.14,15 The equilibrium constant for the normal peptide bond in proteins favors the trans conformation by a factor of 103-104 or so. The peptidyl-prolyl bond is an exception that has some 2-20% of cis isomer in model peptides (see Chapter 1, Figure 1.3). Further, it is often found as the cis isomer in native structures. (Replacement of ds-prolines with other amino acids by protein engineering can retain the cis stereochemistry.16) The interconversion of cis to trans in solution is quite slow, having half-lives of 10-100 s at room temperature and neutral pH. This has two important consequences. First, a protein that has several... 

Although all proteinogenic amino acids form predominantly anti peptide bonds, a search in the Brookhaven Protein Database revealed that approximately 6-7% of all X-prolyl peptide bonds are found in the syn conformation in the native state of proteins [8]. The reason for this relatively frequent occurrence of syn-prolyl peptide bonds lies in steric repulsion of the proline 3 protons and the adjacent N-terminal amino acid in the anti conformation, resulting in a low barrier of rotation and energetically similar syn and anti isomers (Figure 1.2.3). 

In native proteins of known three-dimensional structure about 7% of all prolyl peptide bonds are cis (Stewart et al., 1990 MacArthur and Thornton, 1991). Usually, the conformational state of each peptide bond is clearly defined. It is either cis or trans in every molecule, depending on the structural framework imposed by the folded protein chain. There are a few exceptions to this rule. In the native states of staphylococcal nuclease (Evans et al., 1987), insulin (Higgins et al., 1988), and calbindin (Chazin et al., 1989) cis-trans equilibria at particular Xaa-Pro bonds have been detected in solution by NMR. In staphylococcal nuclease, the cis conformer of the Lys 116-Pro 117 bond can be selectively stabilized by bind-... 

Normally, the native protein, N, has each prolyl peptide bond in a unique arrangement, either cis or trans. After unfolding [N— Up, Eq. (1)], however,... 

Under solvent conditions that strongly favor folded structure ( strongly native conditions ), chains with certain incorrect isomers can rapidly form intermediates, Ig, which are partially nativelike [Eq. (2)] well before prolyl peptide bond reisomerization occurs (Cook et ai, 1979 ... 

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

The fraction of Us molecules depends on the number of proline residues and on their isomeric state in the native protein. In particular, the presence of cts-prolyl peptide bonds in the folded molecules leads to a high fraction of Us, since in unfolded proteins the cis state is populated to a small extent only. Adler and Scheraga (1990) showed by NMR that in heat-unfolded RNase A the nonnative trans isomers predominate at both Pro93 and Proll4. The Up molecules dominate in the unfolded state of proteins that have only tram-prolyl peptide bonds, such as lysozyme (Kato et ai, 1981, 1982), cytochrome c (Ridge el ai, 1981 Nall,... 

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