Enzyme-catalyzed hydrolysis protease

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

Enzyme-catalyzed hydrolysis, exploiting the esterase activity of proteases such as trypsin and chymotrypsint ° l or carboxypeptidase has opened alternative routes to the deprotection of several peptide methyl, ethyl, and ferf-butyl esters. In fact, methyl, ethyl, and benzyl esters are successfully hydrolyzed from protected peptides using the alkaline protease from Bacillus subtilis or alcalase from Bacillus licheniformis which accepts... 

If the enzyme-catalyzed hydrolysis of peptide bond involves a simple reversible reaction as shown by Equation 2.5 then, indeed, the enzyme must catalyze the rate of formation of peptide bond from amino acids (i.e., lq,-step), provided the amino acids do not react irreversibly with the enzyme. Incidentally, if the function of serine proteases is to catalyze both the rate of hydrolytic cleavage and the rate of formation of protein peptide bond, then, probably, these enzymes cannot digest the proteins that we eat and, consequently, the results would have been disastrous for all protein-eating creatures — which certainly Nature will never allow. Although the mechanisms of most of the enzyme-catalyzed reactions are unknown, even at a very rudimentary level, the mechanism of a-chymotrypsin-catalyzed hydrolysis of peptide bond has been relatively well understood. The reaction has been almost ascertained to involve acylation and deacylation of enzyme as shown by Equation 2.6. Widely accepted mechanisms for acylation and deacylation steps are shown in Scheme 2.6 and Scheme 2.7. ... 

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. 

Amide hydrolysis is common in biological chemistry. Just as the hydrolysis of esters is the initial step in the digestion of dietary fats, the hydrolysis of amides is the initial step in the digestion of dietary proteins. The reaction is catalyzed by protease enzymes and occurs by a mechanism almost identical to that we just saw for fat hydrolysis. That is, an initial nucleophilic acyl substitution of an alcohol group in the enzyme on an amide linkage in the protein gives an acyl enzyme intermediate that then undergoes hydrolysis. 

Both alkaline proteases form an intermediate, the acyl-enzyme complex, on the reaction coordinate from the amino acid component to the dipeptide, which is formed by the triad Ser-(or Cys-)-His-Asp (or -Glu) (see Chapter 9, Section 9.5). The acyl-enzyme complex can be formed with the help of an activated amino acid component such as an amino acid ester. The complex can react either with water to the undesired hydrolysis product, the free amino acid, or with the amine of the nucleophile, such as an amino acid ester or amide, to the desired dipeptide. The particular advantage of enzyme-catalyzed peptide synthesis rests in the biocatalyst specificity with respect to particular amino acids in electrophile and nucleophile positions. Figure 7.26 illustrates the principle of kinetically and thermodynamically controlled peptide synthesis while Table 7.3 elucidates the specificity of some common proteases. 

Kurokawa et al81 reported the enzyme-catalyzed kinetic resolution of racemic N-carbamoyl, A-Boc, N-Cbz proline esters and prolinols using protease and Candida antarctica lipase B. The latter was efficient in the enantioselctive hydrolysis of both N-Boc and N-Cbz proline derivatives with E > 100. 

The serine hydrolase family is one of the largest and most diverse classes of enzymes. They include proteases, peptidases, lipases, esterases, and amidases and play important roles in numerous physiological and pathological process including inflammation [53], angiogenesis [54], cancer [55], and diabetes [56]. This enzyme family catalyzes the hydrolysis of ester, thioester, and amide bonds in a variety of protein and nonprotein substrates. This hydrolysis chemistry is accomplished by the activation of a conserved serine residue, which then attacks the substrate carbonyl. The resulting covalent adduct is then cleaved by a water molecule, restoring the serine to its active state [57] (Scheme 1). 

Synthetic peptide inhibitors have been developed for a variety of proteases [199-204]. Peptide inhibitors of the metalloprotease angiotensin I converting enzyme (ACE) are of major importance as hypertensive agents [13, 31]. A variety of peptides derived from protease-catalyzed hydrolysis of com cc-zein [202-203] or of wheat germ protein [199, 204] inhibit ACE (Table 6). The most potent of such plant-derived ACE inhibitory peptides is Ile-Val-Tyr (IVY) (Ki 0.1 xM) [199, 204], Further plant-derived peptide ACE inhibitors include the tripeptide glutathione [73, 82], the glutathione -related peptide Y-L-glutamyl-(+)-allyl-L-cysteine sulphoxide [73, 82, 200, 201] and the tripeptide His-His-Leu (HHL) from fermented soybean [201] (Table 6). 

Recently a simplified process was developed for incorporating l-methionine directly into soy proteins during the papain-catalyzed hydrolysis (21). The covalent attachment of the amino acid requires a very high concentration of protein and occurs through the formation of an acyl-enzyme intermediate and its subsequent aminolysis by the methionine ester added in the medium. From a practical point of view, the main advantage of enzymatic incorporation of amino acids into food proteins, in comparison with chemical methods, probably lies in the fact that racemic amino acid esters such as D,L-methionine ethyl ester can be used since just the L-form of the racemate is used by the stereospecific proteases. On the other hand, papain-catalyzed polymerization of L-methio-nine, which may occur at low protein concentration (39), will result in a loss of methionine because of the formation of insoluble polyamino acid chains greater than 7 units long. 

Proteases are enzymes catalyzing the hydrolysis of peptide bonds. They form one of the largest enzyme families encoded by the human genome, with more than 500 active members. Based on the different catalytic mechanisms of substrate hydrolysis, these enzymes are divided into four major classes serine/threonine, cysteine, metallo, and aspartic proteases. In serine, cysteine, and threonine proteases, the nucleophile of the catalytic site is a side chain of an amino acid in the protease (covalent catalysis). In metallo and aspartic proteases, the nucleophile is a water molecule activated through the interaction with amino acid side chains in the catalytic site (non-covalent catalysis) (Gerhartz et al., 2002). 

A type I thioesterase domain is present at the NHj-terminal of the animal FAS and is responsible for catalyzing hydrolysis of the completed fatty acyl chain from the enzyme. The active site contains both conserved serine and histidine residues [87] and is thought to function via a mechanism similar to that of the serine proteases [50] however, no conserved acidic residue is present to complete the charge relay/transfer. A second variety of thioesterase (type II) is encoded as a separate protein and interacts with the multifunctional FAS to release medium chain fatty acids [88, 89]. This enzyme has a weak sequence similarity to the type I thioesterase, which includes the conserved active site serine and histidine residues. These enzymes are also homologous to proteins encoded by genes involved in the synthesis of peptide antibiotics [90,91] (see below). 

Since the importance of dietary protein for human health and nutrition has long been recognized, it is surprising that remarkably little is known about the physiological processes involved in protein digestion. Many proteases—enzymes catalyzing the hydrolysis of peptide bonds—... 

More than a third of all known proteolytic enzymes are serine proteases (2). The family name stems from the nucleophilic serine residue within the active site, which attacks the carbonyl moiety of the substrate peptide bond to form an acyl-enzyme intermediate. Nucleophilicity of the catalytic serine is commonly dependent on a catalytic triad of aspartic acid, histidine, and serine—commonly referred to as a charge relay system (3). First observed by Blow over 30 years ago in the structure of chymotrypsin (4), the same combination has been found in four other three-dimensional protein folds that catalyze hydrolysis of peptide bonds. Examples of these folds are observed in trypsin. 

Enzymes such as proteases (122), subtilisin (123), acylases, peptidases, amidases, and lipases (124) are reported to catalyze amide bond formation with, in some cases, enantiospeciflcity of over 99%. Despite limited enzyme-substrate compatibility, specific conditions have been developed to reverse their natural reactivity, which is in favor of the hydrolysis. For example, Kyotorphin (Tyr-Arg) (125), a potent analgesic, was produced on an industrial scale using a-chymotrypsin, a peptidase isolated from bovine pancreas. 

Initial attempts to achieve an enzyme-catalyzed deprotection of the carboxy group of peptides centred around the use of the endopeptidases chymotrypsin, trypsin,and thermolysin.P l Thermolysin is a protease obtained from Bacillus thermoproteolyticus that hydrolyzes peptide bonds on the annino side of the hydrophobic amino acid residues (e.g., leucine, isoleucine, valine, phenylalanine). It cleaved the supporting tripeptide ester H-Leu-Gly-Gly-OEt from a protected undecapeptide (pH 7, rt). The octapeptide, thus obtained, is composed exclusively of hydrophilic annino acids. Due to the broad substrate specificity of thermolysin and the resulting possibility of unspecific peptide hydrolysis, this method is of limited application. 

CalriuTin-activated protease in muscle The enzyme catalyzes the hydrolysis of troponir to tropomyosin. The enzyme is thought to be used in the degradatian and turnover of muscle fibers. 

Hydrolases catalyze the addition of water to a substrate by means of a nucleophilic substitution reaction. Hydrolases (hydrolytic enzymes) are the biocatalysts most commonly used in organic synthesis. They have been used to produce intermediates for pharmaceuticals and pesticides, and chiral synthons for asymmetric synthesis. Of particular interest among hydrolases are amidases, proteases, esterases, and lipases. These enzymes catalyze the hydrolysis and formation of ester and amide bonds. 

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