Carboxypeptidase

Carboxypeptidase N (kininase I) cleaves C-terminal lysyl and argininyl residues from peptides. The assay described here is about 1000-fold more sensitive 

The enzyme is assayed by quantifying by HPLC the furylacryloyl-Ala produced from either of the two substrates. The substrate and product are separated on a Cjg RP column (4 gm, Waters) by using 0.1% TFA in 80 20 water-acetonitrile. The eluate is monitored at 280 nm. The formation of product is linear with time and proportional to enzyme concentration when hydrolysis does not exceed 20 to 25%. 

The standard assay used by Grimwood et al. (1988) consisted of 10 fiL 2.0 mAf substrate in 0.05 M Hepes (pH 7.75) and 10 /xL of enzyme. The reactions proceeded at 37°C and were terminated with 10 /xL 0.4 M H3PO4. Samples of 20 /xL were injected. The lysine-containing peptide is cleaved three times more rapidly than the argininyl peptide. A guard column containing the same chromatographic medium was replaced every 2 weeks. 

Applicability of the assay was shown for human carboxypeptidase N purified from plasma as well as enzyme in unpurified plasma and conditioned medium from a human hepatoma cell line (Hep G2). 

SURVEY OF ENZYMATIC ACTIVITIES ASSAYED BY THE HPLC METHOD 

With a single pure polypeptide chain it should theoretically be possible to determine the complete sequence of residues by following the rate of liberation of different amino acids under the action of carboxypeptidase. No such experiments have been described, but it would be interesting to know how far this method could be applied in practice. Clearly it would be impossible to draw any conclusions beyond the C-terminal residues if more than one peptide chain were present, as in the case of insulin. 

A more advanced picture of enzyme action in biosynthesis is given in Chapter 3 when discussing desaturase enzymes. 

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An amino acid sequence is ambiguous unless we know the direction m which to read It—left to right or right to left We need to know which end is the N terminus and which IS the C terminus As we saw m the preceding section carboxypeptidase catalyzed hydrolysis cleaves the C terminal ammo acid and so can be used to identify it What about the N terminus ... 

The shape of a large protein is influenced by many factors including of course Its primary and secondary structure The disulfide bond shown m Figure 27 18 links Cys 138 of carboxypeptidase A to Cys 161 and contributes to the tertiary structure Car boxypeptidase A contains a Zn " ion which is essential to the catalytic activity of the enzyme and its presence influences the tertiary structure The Zn ion lies near the cen ter of the enzyme where it is coordinated to the imidazole nitrogens of two histidine residues (His 69 His 196) and to the carboxylate side chain of Glu 72... 

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... 

FIGURE 27 19 Proposed mechanism of hydrolysis of a peptide catalyzed by carboxypeptidase A The peptide is bound at the active site by an ionic bond between its C terminal ammo acid and the positively charged side chain of arginine 145 Coordination of Zn to oxygen makes the carbon of the carbonyl group more positive and increases the rate of nucleophilic attack by water... 

Living systems contain thousands of different enzymes As we have seen all are structurally quite complex and no sweeping generalizations can be made to include all aspects of enzymic catalysis The case of carboxypeptidase A illustrates one mode of enzyme action the bringing together of reactants and catalytically active functions at the active site... 

Carboxypeptidase catalyzed hydrolysis can be used to identify the C terminal ammo acid The N terminus is determined by chemical means One reagent used for this purpose is Sanger s reagent 1 fluoro 2 4 dimtrobenzene (see Figure 27 9)... 

Metabolic Functions. Zinc is essential for the function of many enzymes, either in the active site, ie, as a nondialyzable component, of numerous metahoenzymes or as a dialyzable activator in various other enzyme systems (91,92). WeU-characterized zinc metahoenzymes are the carboxypeptidases A and B, thermolysin, neutral protease, leucine amino peptidase, carbonic anhydrase, alkaline phosphatase, aldolase (yeast), alcohol... 

Group II consists of the enkephalins which come from the 267-aniino acid piecuisoi pro-enkephalin A [88402-54-4] (Fig. 2). This proteia contains four copies of Met-enkephalin, one copy of Leu-enkephalin, and the extended peptides Met-enkephalin-Arg -Phe (the last Met-enkephalin sequence ia Fig. 2) and Met-enkephalin-Arg -Gly -Leu (the fourth Met-enkephalin sequence ia Fig. 2) (25,26). AH of these products ate formed by trypsin-like cleavage between pairs of basic residues. The extended enkephalin peptides are further cleaved by carboxypeptidase E (27) to form authentic Met-enkephalin. 

Preliminary investigations involving a P-lactam-sensitive, bifimctional D-alanyl-carboxypeptidase—transpeptidase (C Pase—T Pase) from Streptomjces R61 have identified the three-dimensional stmcture and catalytic site of interaction with penicillins (63). 

Enzymes Degrading Macromolecules. Enzymes that degrade macromolecules such as membrane polysaccharides, stmctural and functional proteins, or nucleic acids, have all shown oncolytic activity. Treatment strategies include the treatment of inoperable tumors with pepsin (1) antitumor activity of carboxypeptidase (44) cytotoxicity of ribonudease (45—47) oncolytic activity of neuraminidase (48—52) therapy with neuraminidase of patients with acute myeloid leukemia (53) antitumor activity of proteases (54) and hyaluronidase treatment in the management of human soHd tumors (55). 

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... 

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.)...

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