Enzymes carboxypeptidase, hydrolysis

There are simple reagents that react selectively with the carboxyl terminus of a peptide, but they have not proved as generally useful for analysis of the C-terminal amino acids as has the enzyme carboxypeptidase A. This enzyme catalyzes the hydrolysis of the peptide bond connecting the amino acid with the terminal carboxyl groups to the rest of the peptide. Thus the amino acids at the carboxyl end will be removed one by one through the action of the enzyme. Provided that appropriate corrections are made for different rates of hydrolysis of peptide bonds for different amino acids at the carboxyl end of the peptide, the sequence of up to five or six amino acids in the peptide can be deduced from the order of their release by carboxypeptidase. Thus a sequence such as peptide-Ser-Leu-Tyr could be established by observing that carboxypeptidase releases amino acids from the peptide in the order Tyr, Leu, Ser ... 

The specificity of the acid carboxypeptidase displays the features typical of all pancreatic carboxypeptidases, hydrolysis of the specific substrate R-X-Y between X and Y (R = peptide residue, Z-, Bz-, Ac-). The amino acid in position Y must have a free carboxyl group dipeptides (having free amino group) are not hydrolyzed. The enzyme hydrolyzes most of the a-amino substituted peptides. The carboxypeptidase was inactive on a number of small amides tried at pH 3.0. A peculiarity of its specificity, however, was its inability to hydrolyze the peptide bond of tripeptides tried in the Table 11. 

There are no significant differences between ethyl and methyl esters concerning synthesis or cleavage. Related protocols of the methyl esters (see Section 2.2.1.1.1.1) are, therefore, applied to the ethyl esters. The usefulness of ethyl esters is somewhat limited by the difficulties encountered in their saponification. Hydrolysis with alkali is feasible, but ethyl esters are less sensitive to nucleophilic attack than methyl esters. Aminolysis and hydrazinolysis as well as cleavage of the alkyl-oxygen bond with lithium iodide in pyridineb l proceed several times slower in the case of ethyl esters. Mild enzyme-catalyzed hydrolysis by trypsin and chymotrypsin,t 2° 2 1 or by carboxypeptidase remains an attractive alternative. 

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... 

The crystal structures of several complexes of the metallo enzyme, carboxypeptidase A (CPA)(EC 3.4.17.1), have been examined in considerable detail. The structure of the complex with glycyl tryosine (Gly-Tyr) as been refined to 2.0 A resolution and reveals inter alia interactions between the amide carbonyl oxygen and the catalytically essential zinc, and between the amide nitrogen and the hydroxyl of tryosine-248 (Tyr-248)(Fig. 11). The proposed mechanisms for hydrolysis of peptide and ester bonds by CPA have relied heavily on these crystal structures, but a clear distinction between the possible roles of glutamate-270 (Glu-270) in nucleophilic attack either by general base catalysis (Fig. 11 A) or by covalent any hydride formation (Fig. IIB) remains a major unresolved problem. Indeed, it is not yet certain whether esters and amides are hydrolyzed by CPA via identical mechanisms. 

The limitations noted for aminopeptidases are true for carboxypeptidases as well. The pancreatic enzyme carboxypeptidase A shows low rates in the hydrolysis of peptides with a basic amino acid (arginine or lysine) as the C-terminal residue. Carboxypeptidase B is particularly effective when the same basic residues occupy the C-terminal position. The yeast enzyme carboxypeptidase Y is less specific and therefore more generally applicable, but probably still unsuited for the elucidation of a longer sequence. 

Substrate specificity of P. may be high, e. g. rennet enzyme, carboxypeptidase B, enterokinase, etc., or low, e.g. pronase, pepsin and intracellular proteinases. Often P. are specific for certain amino acid residues, e.g. trypsin catalyses hydrolysis of arginyl and lysyl bonds. Pancreatic P. are among the most extensively studied of all enzymes. 

Enzymes, carboxypeptidases, or amino peptidases have been found that can release C- and N-terminal amino acids selectively. Chromatography is used to separate the product of enzymic or organic hydrolysis. 

The observations that have been summarized immediately suggest structure-function relationship. It seems that a 15-residue segment of the C-terminal polypeptide chain is not essential to activity. Changes in the amino acid sequence can take place without inducing loss of activity. Such a view is supported by degradation experiments. Pepsin degradation of the 39-resi-due polypeptide leads to the formation of three smaller but active polypeptides one of 28, one of 30, and one of 33 amino acid residues. Each of these polypeptides includes the N-terminal serine. In contrast, enzymic (carboxypeptidase, pepsin, chymotrypsin) hydrolysis of the 24 N-terminal polypeptide destroys hormonal activity. Furthermore, the elimination of the single amino acid of the 24 N-terminal peptide seems to inactivate the molecule completely. 

Wulff s group has continued to demonstrate their prowess in this field by more recently reporting the preparation of an artificial model for the natural enzyme carboxypeptidase A (Scheme 24). The catalytic elements to the carbonate hydrolysis consisted of either a Zn + or a Cu + center, and the amidinium group used in previous studies (see above). Both were incorporated in a defined orientation in the transition state-imprinted active site that was facilitated by the inclusion of an intentionally designed tetrahedral TSA. Competitive inhibition experiments with the template indicated that stoichiometric noncovalent imprinting provided a very efficient imprinting strategy. 

Peptides can also be hydrolyzed at specific sites using enzymes. The enzyme carboxypeptidase catalyzes the hydrolysis of the amide bond nearest the C-terminal end, forming the C-terminal amino acid and a peptide with one fewer amino acid. In this way, carboxypeptidase is used to identify the C-tetminal amino acid. 

The enzyme carboxypeptidase catalyses the hydrolysis of polypeptides, and here we consider its inhibition. The following results were obtained when the rate of the enzymolysis of carbobenzoxy-glycyl-D-phenylalanine (CBGP) was monitored without inhibitor ... 

Figure 2.3 The carboxylic acid end of a protein sitting in the active site, a pocket in the enzyme carboxypeptidase, showing how it is held in place by bonding to the Zrf atom and various amino acids. The probable mechanism, based on the hydrolysis of a known peptide, is shown, with the water molecule used for the hydrolysis shown in bold type Once the amino-acid is cleaved, it can diffuse away and the protein moves up into the pocket for the next amino-acid to be cleaved in the same way...

Knowing how the protein chain is folded is a key ingredient m understanding the mechanism by which an enzyme catalyzes a reaction Take carboxypeptidase A for exam pie This enzyme catalyzes the hydrolysis of the peptide bond at the C terminus It is... 

Carboxypeptidases are zinc-containing enzymes that catalyze the hydrolysis of polypeptides at the C-terminal peptide bond. The bovine enzyme form A is a monomeric protein comprising 307 amino acid residues. The structure was determined in the laboratory of William Lipscomb, Harvard University, in 1970 and later refined to 1.5 A resolution. Biochemical and x-ray studies have shown that the zinc atom is essential for catalysis by binding to the carbonyl oxygen of the substrate. This binding weakens the C =0 bond by... 

An artificial metalloenzyme (26) was designed by Breslow et al. 24). It was the first example of a complete artificial enzyme, having a substrate binding cyclodextrin cavity and a Ni2+ ion-chelated nucleophilic group for catalysis. Metalloenzyme (26) behaves a real catalyst, exhibiting turnover, and enhances the rate of hydrolysis of p-nitrophenyl acetate more than 103 fold. The catalytic group of 26 is a -Ni2+ complex which itself is active toward the substrate 1, but not toward such a substrate having no metal ion affinity at a low catalyst concentration. It is appearent that the metal ion in 26 activates the oximate anion by chelation, but not the substrate directly as believed in carboxypeptidase. 

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. 

Procarboxypeptidase A is activated by the removal of a peptide of some 64 residues from the N-terminus by trypsin.153 This zymogen has significant catalytic activity. As well as catalyzing the hydrolysis of small esters and peptides, procarboxypeptidase removes the C-terminal leucine from lysozyme only seven times more slowly than does carboxypeptidase. Also, the zymogen hydrolyzes Bz-Gly-L-Phe with kcsA = 3 s-1 and KM = 2.7 mM, compared with values of 120 s 1 and 1.9 mM for the reaction of the enzyme.154 In contrast to the situation in chymotrypsinogen, the binding site clearly pre-exists in procarboxypeptidase, and the catalytic apparatus must be nearly complete. 

The angiotensin converting enzyme (ACE) is a zinc carboxypeptidase that catalyzes the hydrolysis of the decapeptide angiotension I to the the octapeptide... 

Although zinc, cadmium, and mercury are not members of the so-called main-group elements, their behavior is very similar because of their having complete d orbitals that are not normally used in bonding. By having the filled s orbital outside the closed d shell, they resemble the group IIA elements. Zinc is an essential trace element that plays a role in the function of carboxypeptidase A and carbonic anhydrase enzymes. The first of these enzymes is a catalyst for the hydrolysis of proteins, whereas the second is a catalyst for the equilibrium involving carbon dioxide and carbonate,... 

The kinetic constants for the carboxypeptidase A catalyzed hydrolysis at pH 9 and 25° were /ccal=61 s Km=0.29 mM. In other words, the enzyme afforded a rate enhancement of 11 orders of magnitude (kcJkchem= 4.7xlO11), and a catalytic proficiency of 15 orders of magnitude ((kL.JKm)/... 

Indeed, all three peptides were rather rapidly degraded by rat jejunal homogenates with tm values of 12-32 min. The peptides 6.84 and 6.85 were also hydrolyzed in rat and human jejunal fluid, whereas 6.86 was less sensitive [211], When examined in the presence of purified enzymes, the three peptides showed different reactivities. The peptides 6.84 and 6.85 were very rapidly degraded by chymotrypsin (f1/2 ca. 1 min) and rapidly by trypsin (tm ca. 20 min) [212], Hydrolysis by carboxypeptidase A was almost as fast as... 

These proteolytic enzymes are all endopeptidases, which hydrolyse links in the middle of polypeptide chains. The products of the action of these proteolytic enzymes are a series of peptides of various sizes. These are degraded further by the action of several peptidases (exopeptidases) that remove terminal amino acids. Carboxypeptidases hydrolyse amino acids sequentially from the carboxyl end of peptides. They are secreted by the pancreas in proenzyme form and are each activated by the hydrolysis of one peptide bond, catalysed by trypsin. Aminopeptidases, which are secreted by the absorptive cells of the small intestine, hydrolyse amino acids sequentially from the amino end of peptides. In addition, dipeptidases, which are structurally associated with the glycocalyx of the entero-cytes, hydrolyse dipeptides into their component amino acids. 

This enzyme [EC 3.4.16.4], also known as serine-type D-alanyl-D-alanine carboxypeptidase, catalyzes the hydrolysis of D-alanyl-D-alanine to yield two D-alanine. This enzyme comprises a group of membrane-bound, bacterial enzymes of the peptidase family Sll. They are distinct from the zinc D-alanyl-D-alanine carboxypeptidase [EC 3.4.17.14]. The enzyme also hydrolyzes the D-alanyl-D-alanine peptide bond in the polypeptide of the cell wall. In addition, the enzyme will also catalyze the transpeptidation of peptidyl-alanyl moieties that are A-acetyl-substituents of D-alanine. The protein is inhibited by j8-lactam antibiotics, which acylate the active-site seryl residue. 

This enzyme [EC 3.4.16.5] (also known as serine-type carboxypeptidase I, cathepsin A, carboxypeptidase Y, and lysosomal protective protein) is a member of the peptidase family SIO and catalyzes the hydrolysis of the peptide bond, with broad specificity, located at the C-terminus of a polypeptide. The pH optimum ranges from 4.5 to 6.0. The enzyme is irreversibly inhibited by diisopropyl fluorophosphate and is sensitive to thiolblocking reagents. 

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