Carboxypeptidases, protein hydrolysis

The primary structure of a protein is the sequence of its amino acids and the location of all its disulfide bridges. The N-terminal amino acid of a peptide or protein can be determined with Edman s reagent. The C-terminal amino acid can be identified with carboxypeptidase. Partial hydrolysis hydrolyzes only some of the peptide bonds. An exopeptidase catalyzes the hydrolysis of a peptide bond at the end of a peptide chain. An endopeptidase catalyzes the hydrolysis of a peptide bond that is not at the end of a peptide chain. 

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... 

Carboxypeptidase A catalyses the hydrolysis of the terminal peptide bond in proteins during the process of digestion ... 

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,... 

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. 

The stereochemistry of reactions at zinc atoms has been studied in small molecules (Auf der Heyde and Nassimbeni, 1984) and in proteins (Holmes and Matthews, 1981 Vallee and Auld, 1990a,b). Zinc enzymes include carboxypeptidase A (Quiocho and Lipscomb, 1971 Rees et al., 1983), in which the zinc is coordinated to two histidine nitrogen atoms, two glutamate oxygen atoms, and water (involved in hydrolysis) (Fig. 26). 

The COOH-terminal amino acid of a peptide or protein may be analyzed by either chemical or enzymatic methods. The chemical methods are similar to the procedures for NH2-terminal analysis. COOH-terminal amino acids are identified by hydrazinolysis or are reduced to amino alcohols by lithium borohydride. The modified amino acids are released by acid hydrolysis and identified by chromatography. Both of these chemical methods are difficult, and clear-cut results are not readily obtained. The method of choice is peptide hydrolysis catalyzed by carboxypeptidases A and B. These two enzymes catalyze the hydrolysis of amide bonds at the COOH-terminal end of a peptide (Equation E2.3), since carboxypeptidase action requires the presence of a free a-carboxyl group in the substrate. 

You may have wondered how the proteolytic enzymes such as trypsin, pepsin, chymotrypsin, carboxypeptidase, and others keep from self-destructing by catalyzing their own hydrolysis or by hydrolyzing each other. An interesting feature of the digestive enzymes is that they are produced in an inactive form in the stomach or the pancreas—presumably to protect the different kinds of proteolytic enzymes from attacking each other or other proteins. 

Trypsin, chymotrypsin, and elastase—three members of the serine protease family—catalyze the hydrolysis of proteins at internal peptide bonds adjacent to different types of amino acids. Trypsin prefers lysine or arginine residues chymotrypsin, aromatic side chains and elastase, small, nonpolar residues. Carboxypeptidases A and B, which are not serine proteases, cut the peptide bond at the carboxyl-terminal end of the chain. Carboxypeptidase A preferentially removes aromatic residues carboxypeptidase B, basic residues. (Illustration copyright by Irving Geis. Reprinted by permission.)... 

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. 

Hydrolysis of peptides and proteins in the GI tract can occur luminally, at the brash border and intracellularly. Luminal activity from the pancreatic proteases trypsin, chymotrypsin, elastase and carboxypeptidase A is mainly directed against large dietary proteins. The main enzymatic activity against small bioactive peptides is derived from the bmsh border of the enterocyte. Brash border proteases, such as aminopeptidase A and N, diaminopeptidease IV and Zn-stable Asp-Lys peptidase, preferentially cleave oligopeptides of up to 10 ammo acid residues and are particularly effective in the cleavage of tri- and tetra-peptides. 

Although it is true that abnormal proteins increase with age, most of them are a result of posttranslational changes. An example is the various isoforms of creatine kinase (CK). Here, the major isoenzyme, CK-MM (isoform CK-33), is normally synthesized in the heart and skeletal muscle. However, after its release into the circulation, carboxypeptidase hydrolyzes the terminal lysine from one of the M-peptides to form CK-32. Subsequent hydrolysis of the terminal lysine from the second M-peptide produces the third isoform, CK-3i (W8). Numerous similar posttranslational proteins are produced. Hence, the presence of abnormal proteins per se does not support this aging theory. 

The action of these two pancreatic exopeptidases on synthetic substrates, proteins, and peptides has been reviewed in detail by Neurath (1960). The specificity requirements which were deduced from studies with synthetic peptides have been confirmed by studies with polypeptides. The structural requirements of specific substrates for both types of carboxy-peptidase are analogous except for the nature of the amino acids which contain the free, ionized a-carboxyl group at the terminus of the substrate. Carboxypeptidase B hydrolyzes most rapidly those bonds formed by terminal lysyl and arginyl residues, whereas carboxypeptidase A hydrolyzes terminal bonds formed by a variety of aromatic, neutral, or acidic amino acids. Of the natural amino acids only carboxyl-terminal prolyl residues are resistant to the action of the enzyme. The rate of hydrolysis depends upon the nature of the side chains of the amino acids which form the susceptible bonds. Thus, differences in the rate of hydrolysis of different substrates may vary several thousandfold. The methods for application of these peptidases to hydrolysis of proteins have been discussed in detail by Canfield and Anfinsen (1963). 

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