Hydrolysis of peptide bonds

Hydrolyzed Vegetable Protein. To modify functional properties, vegetable proteins such as those derived from soybean and other oil seeds can be hydrolyzed by acids or enzymes to yield hydrolyzed vegetable proteins (HVP). Hydrolysis of peptide bonds by acids or proteolytic enzymes yields lower molecular weight products useful as food flavorings. However, the protein functionaHties of these hydrolysates may be reduced over those of untreated protein. 

Deamidation of soy and other seed meal proteins by hydrolysis of the amide bond, and minimization of the hydrolysis of peptide bonds, improves functional properties of these products. For example, treatment of soy protein with dilute (0.05 A/) HCl, with or without a cation-exchange resin (Dowex 50) as a catalyst (133), with anions such as bicarbonate, phosphate, or chloride at pH 8.0 (134), or with peptide glutaminase at pH 7.0 (135), improved solubiHty, whipabiHty, water binding, and emulsifying properties. 

Figure 11.4 Serine proteinases catalyze the hydrolysis of peptide bonds within a polypeptide chain. The bond that is cleaved is called the scissile bond. (Ra) and (Rb)j/ represent polypeptide chains of varying lengths.

The most recent advance in treating HIV infections has been to simultaneously attack the virus on a second front using a protease inhibitor. Recall from Section 27.10 that proteases are enzymes that catalyze the hydrolysis of proteins at specific points. When HIV uses a cell s DNA to synthesize its own proteins, the initial product is a long polypeptide that contains several different proteins joined together. To be useful, the individual proteins must be separated from the aggregate by protease-catalyzed hydrolysis of peptide bonds. Protease inhibitors prevent this hydrolysis and, in combination with reverse transcriptase inhibitors, slow the reproduction of HIV. Dramatic reductions in the viral load in HIV-infected patients have been achieved with this approach. 

Protease inhibitor (Section 28.13) A substance that interferes with enzyme-catalyzed hydrolysis of peptide bonds. 

Peptidases are enzymes that catalyse the hydrolysis of peptide bonds - the bonds between amino acids that are found in peptides and proteins. The terms protease , proteinase and proteolytic enzyme are synonymous, but strictly speaking can only be applied to peptidases that hydrolase bonds in proteins. Because there are many peptidases that act only on peptides, the term peptidase is recommended. Peptidases are included in subclass 3.4 of enzyme nomenclature [1,5]. 

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

Chemical, thermal, or enzymatic treatments are required to obtain analysable samples. Two typical methods used to achieve the hydrolysis of peptidic bonds are enzymatic and chemical catalysis [73]. The reaction times for enzymatic hydrolysis are long and typically lie in the range of 4 8 h [47]. Additionally, they demand purification procedures to get rid of the excess enzyme that could interfere in the protein identification. Due to these drawbacks, this method of hydrolysis finds limited use in the conservation science field. 

The formation of crosslinks in silk fibroin increases the tenacity and resistance to deformation of the fibres, as reflected in the initial modulus and the yield point. This protective effect conferred by fixation of the bifunctional dye Cl Reactive Red 194 was not shown by the monofunctional Orange 16, which is unable to form crosslinks. The loss in tenacity of undyed silk that is observed on treatment at 90 °C and pH 7 for 2 hours is attributable to lowering of the degree of polymerisation (DP) by hydrolysis of peptide bonds. The crosslinking action of bifunctional dyes tends to compensate for this loss in DP and provides an intermolecular network that helps to maintain the physical integrity of the fibre structure [124] ... 

Fig. 3.3. Major steps in the hydrolase-catalyzed hydrolysis of peptide bonds, taking chymo-trypsin, a serine hydrolase, as the example. Asp102, His57, and Ser195 represent the catalytic triad the NH groups of Ser195 and Gly193 form the oxyanion hole . Steps a-c acylation Steps d-f deacylation. A possible mechanism for peptide bond synthesis by peptidases is represented by the reverse sequence Steps f-a.

The above examples demonstrate the behavior of peptide bonds at neutral pH. Information is also available on the pH-rate profile of hydrolysis of peptide bonds, as exemplified by N-(phenylacetyl)glycyl-D-valine (6.47), an acyclic penicillin G analogue [69], As a preliminary observation, we note that this compound contains a single stereogenic center, meaning that results obtained with its enantiomer A-(phcnylacctyl)-Gly-Val would have been identical under the achiral conditions of the study. 

Ser-Xaa or Thr-Xaa Hydrolysis of peptide bonds Luteinizing hormone releasing... 

The hydrolysis of peptide bonds catalyzed by the serine proteases has been the reaction most extensively studied by low-temperature trapping experiments. The reasons for this preference are the ease of availability of substrates and purified enzymes, the stability of the proteins to extremes of pH, temperature, and organic solvent, and the existence of a well-characterized covalent acyl-enzyme intermediate. Both amides and esters are substrates for the serine proteases, and a number of chromo-phoric substrates have been synthesized to simplify assay by spectrophotometric techniques. 

Protein digestion occurs in two stages endopeptidases catalyse the hydrolysis of peptide bonds within the protein molecule to form peptides, and the peptides are hydrolysed to form the amino acids by exopeptidases and dipeptidases. Enteropeptidase initiates pro-enzyme activation in the small intestine by catalysing the conversion of trypsinogen into trypsin. Trypsin is able to achieve further activation of trypsinogen, i.e. an autocatalytic process, and also activates chymotrypsinogen and pro-elastase, by the selective hydro-... 

For example, chymotrypsin cleaves peptides on the C-terminal side of aromatic amino acid residues phenylalanine, tyrosine, and tryptophan, and to a lesser extent some other residues with bulky side-chains, e.g. Leu, Met, Asn, Gin. On the other hand, trypsin cleaves peptides on the C-terminal side of the basic residues arginine and lysine. Elastase usually catalyses hydrolysis of peptide bonds on the C-terminal side of neutral aliphatic amino acids, especially glycine or alanine. These three pancreatic enzymes are about 40% identical in their amino acid sequences, and their catalytic mechanisms are nearly identical. 

This enzyme [EC 3.4.22.17] is an intracellular, nonlyso-somal member of the peptidase family C2. The enzyme catalyzes the calcium ion-dependent hydrolysis of peptide bonds with preference for Tyr-Xaa, Met-Xaa, or Arg-Xaa with a leucyl or valyl residue at the P2 position. There are two main types of calpain. One has a high calcium sensitivity in the micromolar range and is called (,-calpain or calpain I. The other calpain has a low calcium sensitivity in the millimolar range and is called m-calpain or calpain II. Forms of calpain exhibiting intermediate calcium sensitivity also exist. 

This zinc-dependent enzyme [EC 3.4.17.1], a member of the peptidase family M14, catalyzes the hydrolysis of peptide bonds at the C-terminus of polypeptides. Little hydrolytic action occurs if the C-terminal amino acid is aspartate, glutamate, arginine, lysine, or proline. Car-boxypeptidase A is formed from a precursor protein, procarboxypeptidase A. 

This enzyme [EC 3.4.21.39], also referred to as mast cell protease I and skeletal muscle (SK) protease, is an endopeptidase that has been isolated from mast cell granules. It belongs to the peptidase family SI and catalyzes the hydrolysis of peptide bonds, preferring Phe-Xaa > Tyr-Xaa > Trp-Xaa > Leu-Xaa. 

This zinc-dependent enzyme [EC 3.4.24.15], also referred to as thimet oligopeptidase and soluble metalloendopep-tidase, catalyzes the hydrolysis of peptide bonds with a preferential cleavage at positions with hydrophobic residues at PI, P2, and P3 and a small amino acid residue at PI. Substrates for this enzyme contain five to fifteen amino acid residues. 

The following articles describe some of the molecular and physical properties of this enzyme [EC 3.4.22.3] which catalyzes the hydrolysis of peptide bonds and exhibits a broad specificity (at Lys-, Ala-, Tyr-, Gly-, Asn-, Leu-, and Val-). 

This calcium-activated enzyme [EC 3.4.21.75] catalyzes the hydrolysis of peptide bonds in protein precursors that results in the release of mature proteins from their proproteins by hydrolysis of ArgXaaYaaArg—Zaa bonds, where Xaa can be any amino acid and Yaa is an arginyl or a lysyl residue. Albumin, complement component C3, and von Willebrand factor are thus released from their respective precursors. Furin is a member of the peptidase family S8. 

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 enzyme [EC 3.4.22.25] catalyzes the hydrolysis of peptide bonds with a preference for Gly-Xaa in proteins and small molecule substrates. The enzyme, a member of the peptidase family Cl, is isolated from the papaya plant, Carica papaya. It is not inhibited by chicken cys-tatin, unlike most other homologs of papain. 

Plasma kallikrein [EC 3.4.21.34], also known as kinino-genin and serum kallikrein, catalyzes the hydrolysis of Arg—Xaa and Lys—Xaa bonds in polypeptides. This includes the Lys—Arg and Arg—Ser bonds in human kininogen, thus producing bradykinin. Tissue kallikrein [EC 3.4.21.35] catalyzes the hydrolysis of peptide bonds, preferentially Arg—Xaa, in smaU-molecule substrates. It catalyzes the breaking of the appropriate bonds in kininogen resulting in the release of lysyl-bradykinin. 

This calcium-ion-activated enzyme [EC 3.4.21.61] catalyzes the hydrolysis of peptide bonds at LysArg—Xaa and ArgArg—Xaa to process yeast a-factor pheromone and killer toxin precursors. 

This enzyme [EC 3.4.22.34] catalyzes the hydrolysis of peptide bonds in proteins (for example, in azocasein) and polypeptides at Asn-Xaa, the preferential bond. 

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