Cleavage of Specific Peptide Bonds

Direct sequencing can only be achieved for peptides no longer than about 50 residues. Beyond this length, results become unreliable due to incomplete reactions and the accumulation of impurities from side reactions. Hence, most proteins must be cleaved into smaller fragments prior to sequencing. A number of chemical and enzymatic reactions are available that break peptide bonds within the chain at specific places. 

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Proteolytic enzymes have been used extensively in the study of protein structui e. First, of course, purified proteases have been employed to catalyze the cleavage of specific peptide bonds in proteins in the process of establishing amino acid sequences and distributions. Within the last few years it has also become apparent that proteolytic enzymes can be utilized to obtain at least semiquantitative information about protein (and nucleic acid) configuration (or secondary-tertiary structure) as well as about amino acid sequence. This use of proteases can be divided into two general areas ... 

A zymogen is converted to the active form by the irreversible cleavage of specific peptide bonds in the protein. 

Table 27.3 Cleavage of Specific Peptide Bonds Catalyzed by Trypsin and Chymotrypsin...

Fig. 3. Intein-mediated protein ligation. The IMPACT system allows affinity purification of proteins fused to an intein-CBD tag and their further isolation with a C-terminal thioester moiety (A), or an N-terminal cysteine (B). (A), N-terminal intein splicing for thioester isolation. Target protein (protein 1) is expressed in E. coli with C-terminally located intein-CBD tag. After specific binding to the chitin resin, the thiol reagent provokes the cleavage of the peptide bond between the target protein and the intein. Whereas the intein-CBD tag remains bound to the chitin resin, the protein thioester is eluted from the column. (B), C-terminal cleavage to obtain N-terminally...

Where peptide chemistry can make a contribution toward the proof of protein structure is in the application and continued formulation of degrada-tive techniques. Since fragmentation of the protein molecule to smaller peptides is probably the key reaction in degradative processes, the search for specific reagents for the selective cleavage of various peptide bonds and the standardization of existing techniques is of prime importance (Katsoyannis, 1961). 

Since in peptides and proteins alkyl halides at pH 2.8 react only with the sulfur of methionine (Gundlach et al, 1959b), this procedure permits specific chemical cleavage of methionyl peptide bonds. 

The exploitation of the esterase activities of chymotrypsin and trypsin opened routes to the hydrolysis of several peptide methyl and ethyl esters at pH 6.4-8 and room temperature. The transformation is not only successful with peptides carrying the respective enzyme-specific amino acids at the C-terminus, but in several examples different amino acids are tolerated at this position. However, a severe drawback of this approach is that numerous peptides are poor substrates or not accepted at all. Moreover, a competitive cleavage of the peptide bond occurs if the peptides contain trypsin- or chymotrypsin-labUe sequences. Therefore, these proteases are not generally useful for a safe C-terminal deprotection. 

Partial acid hydrolysis is generally non-specific but can still be exploited for the cleavage of the peptide bond at aspartyl residues. The Asp-Pro linkage is exceptionally sensitive to acids and is cleaved by dilute hydrochloric acid at room temperature. At elevated temperatures very weak acids such as 0.03 M... 

Many other difficulties arise in specific cases of solid-phase peptide synthesis, e.g., destruction of the tryptophan residue during acidolytic cleavage of resin-peptide bond, pyroglutamyl derivative formation, chain termination by acetylation with acetic acid leaching from teflon components, and formation of y-glutamyl peptides. Solutions to these problems provide the basis for much of the continuing work in the field. 

Atassi, M. Z. Specific Cleavage of Tryptophyl Peptide Bonds with Periodate in Sperm Whale Myoglobin. Arch. Biochem. Biophys. 120, 56-59 (1967). 

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

The best way to show you t>ow the overlap method of peptide sequencing works is by a specific example. In this example, we will illustrate the use of the two most commonly used enzymes for selective peptide cleavage. One is trypsin, a proteolytic enzyme of the pancreas (MW 24,000) that selectively catalyzes the hydrolysis of the peptide bonds of basic amino acids, lysine and... 

The inactive precursors are called trypsinogen, pepsinogen, chymotrypsino-gen, and procarboxypeptidase. These precursors are converted to the active enzymes by hydrolytic cleavage of a few specific peptide bonds under the influence of other enzymes (trypsin, for example, converts chymotrypsinogen to chymotrypsin). The digestive enzymes do not appear to self-destruct, probably because they are so constructed that it is sterically impossible to fit a part of one enzyme molecule into the active site of another. In this connection, it is significant that chymotrypsin attacks denatured proteins more rapidly than natural proteins with their compact structures of precisely folded chains. 

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