Enzyme carboxypeptidases

Figure 4.19 Schematic and topological diagrams for the structure of the enzyme carboxypeptidase. The central region of the mixed p sheet contains four adjacent parallel p strands (numbers 8, 5, 3, and 4), where the strand order is reversed between strands 5 and 3. The active-site zinc atom (yellow circle) is bound to side chains in the loop regions outside the carboxy ends of these two p strands. The first part of the polypeptide chain is red, followed by green, blue, and brown. (Adapted from J. Richardson.)...

Analogous techniques facilitating sequencing from a polypeptide s C-terminus remain to be satisfactorily developed. The enzyme carboxypeptidase C sequentially removes amino acids from the C-terminus, but often only removes the first few such amino acids. Furthermore, the rate at which it hydrolyses bonds can vary, depending on what amino acids have contributed to bond formation. Chemical approaches based on principles similar to the Edman procedure have been attempted. However, poor yields of derivatized product and the occurrence of side reactions have prevented widespread acceptance of this method. 

The enzyme carboxypeptidase A is particularly amenable to structural investigation crystal structures of the enzyme, of complexes of the enzyme with substrates, substrate analogues and inhibitors, and of transition-state analogues are available. To isolate an enzyme-substrate complex for a one-substrate enzyme reaction, or for an enzyme reaction where water is a... 

Model L The Use of Synthetic Y Zeolites as Models for the Migration of Zn2+ Ions in the Enzyme Carboxypeptidase A... 

Figure 2-14 A "ribbon" drawing of the 307- residue proteinhydrolyzing enzyme carboxypeptidase A. In this type of drawing wide ribbons are used to show 3 strands and helical turns while narrower ribbons are used for bends and loops of the peptide chains. The direction from the N terminus to C terminus is indicated by the arrowheads on the 3 strands. No individual atoms are shown and side chains are omitted. Courtesy of Jane Richardson.117...

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 ... 

Chemical methods for carboxyl end-group determination are considerably less satisfactory. Treatment of the peptide with anhydrous hydrazine at 100°C results in conversion of all the amino acid residues to amino acid hydrazides except for the carboxyl-terminal residue, which remains as the free amino acid and can be isolated and identified chro-matographically. Alternatively, the polypeptide can be subjected to limited breakdown (proteolysis) with the enzyme carboxypeptidase. This results in release of the carboxyl-terminal amino acid as the major free amino acid reaction product. The amino acid type can then be identified chroma-tographically. 

Matrix-assisted laser desorption ionization is another ionization mode used for MS analysis. Enzymatically digested peptides have been studied using a 90-well microchip constmcted in a MALDI plate format (see Figure 7.41). Peptide digestion was initiated in the MALDI interface where the peptide hormone, adreno-corticotropin (ACTH) was mixed with the enzyme carboxypeptidase Y. The mixing process was self-activated in the vacuum conditions. Subsequent TOF MS analysis produced kinetic information of the peptide digestion reaction [820]. 

Requirements of Active Sites in Enzymes Carboxypeptidase, which sequentially removes carboxyl-terminal amino acid residues from its peptide substrates, is a single polypeptide of 307 amino acids. The two essential catalytic groups in the active site are furnished by Arg145 and Glu270. 

Alignment of five completely sequenced members of the pancreatic digestive enzyme carboxypeptidase family1 is shown in Fig. 1, along with hepatopancreatic carboxypeptidase B from crayfish.8 Only the amino acid... 

There is no efficient method for sequencing several amino acids of a peptide starting from the C terminus. In many cases, however, the C-terminal amino acid can be identified using the enzyme carboxypeptidase, which cleaves the C-terminal peptide bond. The products are the free C-terminal amino acid and a shortened peptide. Further reaction cleaves the second amino acid that has now become the new C terminus of the shortened peptide. Eventually, the entire peptide is hydrolyzed to its individual amino acids. 

The enzyme carboxypeptidase A hydrolyzes amino acids from the C-terminal end of peptides, provided that the C-terminal residue is not proline, lysine, or arginine. Fig. 4-13 shows the sequential release of amino acids from a protein by means of treatment with carboxypeptidase A. Deduce the sequence at the C terminus. 

The structure of the enzyme carboxypeptidase-A, which contains 307 amino acids. 

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 most successful method of determining the C-terminal residue has been enzymatic rather than chemical. The C-terminal residue is removed selectively by the enzyme carboxypeptidase (obtained from the pancreaa), which cleaves only peptide linkages adjacent to free a/p/ra-carboxyl groups in polypeptide chains. The analysis can be repeated on the shortened peptide and the ney C-terminal residue identified, and so on. 

The Edman degradation is an excellent method of analysis for the N-ter-minal residue, but a complementary method of analysis for the C-terminal residue is also valuable. The best method currently available uses the enzyme carboxypeptidase to cleave the C-terminal amide bond in a peptide chain. 

Metalloprotease inhibitors - also known as metalloproteases or zinc proteases - are proteolytic enzymes of which the activity depends on metal ions, normally bound Zn -. Examples of metalloproteases are the pancreatic enzymes carboxypeptidase A and B, elastase, the well-characterized bacterial enzyme thermolysin and the collagenase family (found in both bacterial and mammalian cells, fibroblast collagenase, neutrophil elastase, gelatinase). 

Immobilization of Enzymes. Enzymes (carboxypeptidase A, B, and Y, chymotrypsin, thermolysin, trypsin, and V -protease), obtained from Sigma were applied directly for immobilization. About 20 mg of each enzyme was dissolved into 0.1N phosphate buffer pH 7.0, and placed into a 10 x 75 mm test tube with 1 g of succinylamidopropyl glass beads. After degassing, 0.02 umole of l-ethyl-3-(3-dimethylamlnopropyl)-carbodilmide (EDC) (Sigma Chemical Co.) was added to the tube which then was sealed with paraffin and rotated at 4°C overnight for simultaneous activation/immobilization. 

A limited amount of information can be obtained by the use of proteolytic enzymes that detach either amino acids or dipeptides sequentially from the C-terminus. They are thus complementary to the aminopeptidases and dipeptidyl aminopeptidases. Two pancreatic enzymes, carboxypeptidases A and B, differ in specificity. The former preferentially liberates C-terminal amino acids with aromatic side chains, somewhat less readily amino acids with alkyl side chains and, more slowly still, other amino acids, but not Pro, Arg, Lys and His. In contrast, carboxypeptidase B releases only C-terminal Arg, Lys and His. Carboxypeptidase Y is much less specific and is capable of removing all amino acids, although Gly and Pro are liberated only slowly. As with aminopeptidases, it is advisable to analyse the hydrolysate at intervals in order to determine the C-terminal sequence of amino acids. An interesting recent development (Carles et al., 1988) uses carboxypeptidase to effect transpeptidation between the protein being sequenced and a tritiated amino acid. The labelled protein is then degraded by various specific methods and then the labelled fragments are isolated by gel electrophoresis and subjected to Edman degradation. 

The C-terminal residue is determined by the use of either a chemical reagent or the enzyme carboxypeptidase. The... 

The Sequazyme C-peptide sequencing kit enables peptide digestion followed by analysis of sequentially truncated peptides using MALDI-TOF-MS. Before digesting and analyzing unknown samples of the peptide (adrenocorticotropic hormone), the activity of the enzyme carboxypeptidase Y (CPY) was checked by dilution experiments with ammonium citrate buffer (pH = 6.1), CPY dilution and the peptide standard angiotensin I. The adrenocorticotropin ACTH (18-39) fragment (0.3 pL) was transferred as a solution of 5 x 10 5M in a 1 1... 

---