Carboxypeptidase, 508 Table

Proteins are hydrolytically cleaved at the peptide linkages by proteases (peptidases, EC3.4.-.-) (Beynon and Bond, 2001 Sterchi and Stdkenn, 1999). Two classes of peptidases are endopeptidases, which cleave internal bonds (e.g. chymotrypsin, trypsin), and exopeptidases, which hydrolyze the terminal residue of a polypeptide chain (e.g. aminopeptidases, carboxypeptidases). Table 12.10 lists some of the common proteases classified mechanistically according to their characteristics. 

The effects of various enzymes on the activity of HPLC fractions that inhibited 3H-PCP binding were investigated. As shown in table 1, pronase (0.5 pg/ml), carboxypeptidase A (0.1 unit/ml), and trypsin (3.0 g/ml ) markedly decreased the potency of 10 n units of PCP-like activity. No significant change in activity was. seen when fractions were incubated with alpha-chymotrypsin. 

Table 12. Specificity of heavy atom binding to carboxypeptidase (after W. N. Lipscombe)...

Table 6.3. Substrate Selectivity of Human Carboxypeptidases A1 andA2 (hCPAl and hCPA2) and Artificial Mutants toward Methotrexate Prodrugs for Use in ADEPT [62] ...

An example of a pseudopeptide containing the CH2-NH group is afforded by N/t9,CH2-NH brady kinin. This analogue was stabilized not only against carboxypeptidase, which cleaves bradykinin at the 8,9-position, but also against ACE, which cleaves it at the 7,8-position (see Table 6.6). 

Zinc may function to promote the nucleophilicity of a bound solvent molecule in both small-molecule and protein systems. The p/Ca of metal-free H2O is 15.7, and the p/Ca of hexaaquo-zinc, Zn (OH2)6. is about 10 (Woolley, 1975) (Table III). In a novel small-molecule complex the coordination of H2O to a four-coordinate zinc ion reduces the to about 7 (Groves and Olson, 1985) (Fig. 2). This example is particularly noteworthy since it has a zinc-bound solvent molecule sterically constrained to attack a nearby amide carbonyl group as such, it provides a model for the carboxypeptidase A mechanism (see Section IV,B). To be sure, the zinc ligands play an important role in modulating the chemical function of the metal ion in biological systems and their mimics. 

Table 2.2.3.3 Determination of the carboxypeptidase and esterase side-activities of Si HNL in comparison with carboxypeptidase II from wheat. ...

II, DJ-I, HSC 54, carboxypeptidase A I, carboxypeptidase A2, and neuropolypeptide h3 decreased (36). Table 3 shows the summary of the proteins whose expression was different between pancreatic cancer tissues and non-cancerous tissues. 

The considerable detail to which we now can understand enzyme catalysis is well illustrated by what is known about the action of carboxypeptidase A. This enzyme (Section 25-7B and Table 25-3) is one of the digestive enzymes of the pancreas that specifically hydrolyze peptide bonds at the C-terminal end. Both the amino-acid sequence and the three-dimensional structure of carboxypeptidase A are known. The enzyme is a single chain of 307 amino-acid residues. The chain has regions where it is associated as an a helix and others where it is associated as a /3-pIeated sheet. The prosthetic group is a zinc ion bound to three specific amino acids and one water molecule near the surface of the molecule. The amino acids bound to zinc are His 69, His 196, and Glu 72 the numbering refers to the position of the amino acid along the chain, with the amino acid at the /V-terminus being number l. The zinc ion is essential for the activity of the enzyme and is implicated, therefore, as part of the active site. 

Carboxypeptidase A was the first metalloenzyme where the functional requirement of zinc was clearly demonstrated (9, 92). In similarity to carbonic anhydrase, the chelating site can combine with a variety of metal ions (93), but the activation specificity is broader. Some metal ions, Pb2+, Cd2+ and Hg2+, yield only esterase activity but fail to restore the peptidase activity. Of a variety of cations tested, only Cu2+ gives a completely inactive enzyme. In the standard peptidase assay, cobalt carboxypeptidase is the most active metal derivative, while it has about the same esterase activity as the native enzyme ((93, 94), Table 6). Kinetically, the Co(II) enzyme shows the same qualitative features as the native enzyme (95), and the quantitative differences are not restricted to a single kinetic parameter. 

Table 7. Apparent stability constants for metal binding in procarboxypeptidase A, carboxypeptidase A and human carbonic anhydrase B...

Metal binding in procarboxypeptidase A is weaker than in the active enzyme ( 107), Table 7). It was proposed that the bonding involves sulfur and a weaker ligand than N (107). In view of the present concept of the chelating site in carboxypeptidase, further studies of the zymogen are necessary. In that connection, the cobalt complex should be valuable. 

In Table II are shown the results from kinetic studies with commercially available gastric and pancreatic enzymes. Trypsin was strongly inhibited, at least at a low concentration of casein as substrate. The hydrolysis of benzoyl arginine ethyl ester (BAEE) by trypsin was non-competitively inhibited, giving a 30% reduction of Vmax at 0.5 mg/ml of the LMW fraction. Carboxypepti-dase A, and to a lesser extent carboxypeptidase B, were non-competitively inhibited as well. Pepsin and chymotrypsin were not affected by the conditions used in these assays. 

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. 

Table 11. Comparative rate of hydrolysis for Aspergillus acid carboxypeptidase on a...

The acid carboxypeptidase from A. saitoi releases the carboxyterminal phenenylalanine-amide (-Phe-NH2) from the carboxy-terminal amidated peptides, such as gastrin-related peptide (/-amyloxycarbonyl (Aoc)-Trp-Met-Asp-Phe-NH2, Aoc-WMDF-NH2) and molluscan cardioexcitatoiy neuropeptide (Phe-Met-Arg-Phe-NH2, FMRF-NH2) [86], The summarized data are shown in Table 12. When gastrin-related peptide was used as a substrate, the enzyme acted only as a carboxyamidase, because of the presence of the hydrophobic amino acid residue, tryptophan, in the P3 [12] position. 

Table 12. Hydrophobicity of side chains of amino acid residue adjacent to carboxyterminal bond to be split by the carboxypeptidase from A. saitoi...

Table 14. Secondary structures of native and deglycosylated acid carboxypeptidases...

Table 15. Kinetic parameters of active and deglycosylated acid carboxypeptidases from Aspergillus saitoi toward Z-Glu-Tyr at pH 3.1 and 30°C...

In their early efforts to apply the by-product design to ACE, Patchett and Maycock had synthesized only succinylproline, based on the findings of Byers and Wolfenden that benzylsuccinate was superior to ben-zylglutamate as an inhibitor of carboxypeptidase A. Similar active site topologies were assumed for these two enzymes. Evidently this was not the case, since Cushman et al. reported in 1977 (96) that glutarylpro-line (compound 6 Table II.) was more active than compound 5 (Table... 

Several plant proteins have been isolated that inhibit the metalloprotease carboxypeptidase A [205-217] (Table 7), notably potato carboxypeptidase inhibitor PCI [207-217] (Table 7). PCI is a small, cysteine-rich protein with a compact knotted structure determined by 3 disulphide links. The C-terminal region inserts into the active site of the carboxypeptidase. The C-terminal glycine is cleaved and remains trapped in the active site, this representing an example of suicide inactivation [207-216]. 

Table 7. Carboxypeptidase protease inhibitor proteins from plants...

Table V) and there are carboxypeptidases that also belong to both families. Substrate specificity alone is not sufficient to rationally design an inhibitor for a new protease since it tells nothing about the active-site functional groups.

Hydrogen-bond partners for internal water molecules. In the five proteins lysozyme, carboxypeptidase, cytochrome c, actinidin and penicillopepsin (Table 19.1), the protein groups (numbers in parentheses) which are bonded to internal water molecules are the main-chain C=0 (75), N-H (38), and side-chain atoms... 

---