Peptide bond enzymatic cleavage

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

PARACEST agents with a response to enzymatic activity have also been reported. Pagel et al. used a Tm1 DOTA monoamide complex containing a peptide chain which is hydrolyzed by the Caspase-3 enzyme (204,205). Following enzymatic cleavage, the PARACEST effect originating from the amide proton disappears due to the hydrolysis of the amide bond. 

Substitution of the l-amino acids with their D-amino acid configurational analogue, will enhance resistance to enzymatic cleavage of peptide bonds. The ll isomer of cyclo(Leu-Leu) hydrolyzes 3.5 times faster than the DL isomer in the presence of 0.5 moll HCl. This difference was explained by both steric shielding ui-trans isomer) and steric strain (ll- j isomer). ... 

Breaking Disulfide Bonds Disulfide bonds interfere with the sequencing procedure. A cystine residue (Fig. 3-7) that has one of its peptide bonds cleaved by the Edman procedure may remain attached to another polypeptide strand via its disulfide bond. Disulfide bonds also interfere with the enzymatic or chemical cleavage of the polypeptide. Two approaches to irreversible breakage of disulfide bonds are outlined in Figure 3-26. 

Enzymatic cleavage of the thioester bond (located either in the middle or at the C-terminus of the peptide) produces a free thiol compound that can be easily monitored by very sensitive assays (e.g., Ellman s reagent). 64 ... 

Once the protein s primary sequence has been determined, the location of disulfide bonds in the intact protein can be established by repeating a specific enzymatic cleavage on another sample of the same protein in which the disulfide bonds have not previously been cleaved. Separation of the resulting peptides shows the appearance of one new peptide and the disappearance of two other peptides, when compared with the enzymatic digestion product of the material whose disulfide bonds have first been chemically cleaved. In fact, these difference techniques are generally useful in the detection of sites of mutations in protein mole-... 

The bond cleavage detection was validated using the enzymatic cleavage of peptides by a-chymotrypsin, and the important features for the success of the... 

A useful method to assay many hydrolytic enzymes is to follow enzymatic activity by determining production or uptake of hydrogen ions during the reaction. For example, consider a protease that catalyzes cleavage of internal peptide bonds in a protein ... 

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