Carboxypeptidase reaction catalyzed

D-Alanine carboxypeptidases (CPases) catalyze removal of the terminal D-alanyl residue from uncross-linked pentapeptides, and the resultant tetrapeptide cannot serve as substrate for transpeptidation. In this way, CPases may regulate the degree of cross-linking. Some CPases catalyze transpeptidation reactions between side... 

The names of most enzymes end in ase, and the enzyme s name tells you something about the reaction it catalyzes. For example, carboxypeptidase A catalyzes the hydrolysis of the C-terminal (carboxy-terminal) peptide bond in polypeptides, releasing the terminal amino acid (Section 22.13). 

A metal ion cofactor acts as a Lewis acid in a variety of ways to help an enzyme catalyze a reaction. It can help bind the substrate to the active site of the enzyme it can complex with the substrate to increase its reactivity it can coordinate with groups on the enzyme, causing them to align in a way that facilitates the reaction and it can increase the nucleophilicity of water at the active site (Section 23.5). We have seen that Zn plays an important role in the hydrolysis reaction catalyzed by carboxypeptidase A (see page 1116). 

Mortensen, U. H., Stennicke, H. R., Raaschou-Nielsen, M., Breddam, K. (1994). Mechanistic study on carboxypeptidase Y-catalyzed transacylation reactions. Mutationally altered enzymes for peptide synthesis.. Am. Chem. Soc., 116,34-41. 

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... 

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. 

One of the most important metals with regard to its role in enzyme chemistry is zinc. There are several significant enzymes that contain the metal, among which are carboxypeptidase A and B, alkaline phosphatase, alcohol dehydrogenase, aldolase, and carbonic anhydrase. Although most of these enzymes are involved in catalyzing biochemical reactions, carbonic anhydrase is involved in a process that is inorganic in nature. That reaction can be shown as... 

The evolutionary classification has a rational basis, since, to date, the catalytic mechanisms for most peptidases have been established, and the elucidation of their amino acid sequences is progressing rapidly. This classification has the major advantage of fitting well with the catalytic types, but allows no prediction about the types of reaction being catalyzed. For example, some families contain endo- and exopeptidases, e.g., SB-S8, SC-S9 and CA-Cl. Other families exhibit a single type of specificity, e.g., all families in clan MB are endopeptidases, family MC-M14 is almost exclusively composed of carboxypeptidases, and family MF-M17 is composed of aminopeptidases. Furthermore, the same enzyme specificity can sometimes be found in more than one family, e.g., D-Ala-D-Ala carboxypeptidases are found in four different families (SE-S11, SE-S12, SE-S13, and MD-M15). 

Specific ester substrates are also hydrolyzed with carboxypeptidase A. For instance, Makinen, Fukuyama, and Kuo (27) have recently studied the enzymic hydrolysis of 0-(trans-p-ch1orocinnamoyl)-L-B-phenyl actate (CICPI.) (47),and the spin labeled nitroxide ester substrate 0-3-(2,2,5,5-tetramethylpyrrol-inyl-l-oxyl)-propen-2-oyl-L-B-phenyllactate (TEPOPL) (48). They have shown that these reactions take place via the formation of a covalent intermediate (the mixed anhydride) which can be isolated under subzero temperature conditions. The hydrolysis of CICPL and TEPOPL catalyzed by carboxypeptidase A is consequently governed by the rate-limiting breaking of the acyl-enzyme. 

In comparison with the marked advancement in sequence determination of the amino terminal region of protein, no good methods have been developed for the determination of the amino acid sequence of the carboxyl terminal region, although several enzymatic and chemical procedures are available. Exoproteinases such as carboxypeptidases A, B, C and Y, have been employed for enzymatic determination. These enzymes catalyze sequential liberation of an amino acid from a carboxyl terminal. However, the reaction efficiency, i.e. velocity and specificity, is not always consistent for all amino acids consisiting of a... 

Because only 40 to 60 amino acid residues can be determined by the Edman procedure, additional methods are needed for larger proteins. Determination of the C-terminal amino acid can be accomplished by treating the protein with carboxypeptidase. This enzyme selectively catalyzes the hydrolysis of the C-terminal amino acid. After the first amino acid has been removed, the enzyme begins to cleave the second amino acid, and so forth. By following the rates at which the amino acids appear, it is possible to determine the first few amino acids at the C-terminal end of the protein by employing this enzyme. However, because the enzyme hydrolyzes different peptide bonds at different rates, it is possible to identify only a few amino acids before the reaction mixture becomes too complex. 

This complex has been shown to be an excellent structural and functional model for the zinc hydrolytic enzymes, particularly carbonic anhydrase but also carboxypeptidase and the zinc phosphate esterases (24-26). The same complex also catalyzes the hydration of acetaldehyde and hydrolysis of carboxylic esters. These reactions appear to progress via a mechanism similar to that proposed for carbonic anhydrase. The rates are slower for [Zn([12]aneN3)OH] than for the enzyme but an order of magnitude faster than for existing model systems such as [(NH3)5Co(OH)]2+ (26). 

The metalloproteases constitute the final major class of peptide-cleaving enzymes. The active site of such a protein contains a bound metal ion, almost always zinc, that activates a water molecule to act as a nucleophile to attack the peptide carbonyl group. The bacterial enzyme thermolysin and the digestive enzyme carboxypeptidase A are classic examples of the zinc proteases. Thermolysin, but not carboxypeptidase A, is a member of a large and diverse family of homologous zinc proteases that includes the matrix metalloproteases, enzymes that catalyze the reactions in tissue remodeling and degradation. 

The kinetics of action of carboxypeptidase are very complex. The pH-rate profile for CPA-catalyzed hydrolysis of peptides is bell shaped, with apparent pK values of about 6.5 and 7.5. The former pK value could be attributed to Glu-270. The pH dependency of the kinetics of the CPA-catalyzed enolization of the ketonic substrate (R)-2-benzyl-3-(p-methoxybenzoyl)pro-pionic acid leads to the establishment (with minimum complications from this relatively simple reaction) of a pK value of 6.03 for the Co" and Zn" CPA, which is probably due to Glu-270. The binding of the substrate to both Zn" and Co" CPA appears to depend on an enzyme-bound group with pXa = 7.56 and 8.29 respectively, and on a group with pXa>9. These are attributed to the ionization of Tyr-248 and the bound water molecule. 

The mechanism by which an enzyme catalyzes its specific reaction is known in varying levels of detail in a number of cases, for example, rihonuclease, lysozyme, serine proteases, carboxypeptidase, aspartate amino transferase, and lactate dehydrogenase. The mechanisms differ, but some useful general principles have emerged from studies of individual enzymes. Enzymes use the normal principles of organic chemistry. 

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