Carboxypeptidases specificity

It is also possible to determine the amino acid at the other end of a peptide chain, the carboxy terminus. The method of choice uses an enzymatic reaction in which one of a number of carboxypeptidases specifically cleaves the terminal amino acid at the carboxy end of the chain. As each carboxy terminal amino acid reacts with the enzyme, a new amino acid is revealed to be cleaved in turn by more carboxypeptidase. Careful monitoring of the production of amino acids as a function of time can g ve a good idea of the sequence (Fig. 23.33). Stih, it is clear that things will get messy as long peptides are turned into a soup containing an increasingly complex mixture of amino acids. 

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

Culley FJ, Brown A, Conroy DM et al (2000) Eotaxin is specifically cleaved by hookworm metal-loproteases preventing its action in vitro and in vivo. J Immunol 165 6447-6453 Davis DA, Singer KE, De La Luz Sierra M et al (2005) Identification of carboxypeptidase N as an enzyme responsible for C-terminal cleavage of stromal cell-derived factor-lalpha in the circulation. Blood 105 4561 568... 

The introduction of redox activity through a Co11 center in place of redox-inactive Zn11 can be revealing. Carboxypeptidase B (another Zn enzyme) and its Co-substituted derivative were oxidized by the active-site-selective m-chloroperbenzoic acid.1209 In the Co-substituted oxidized (Co111) enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Oxidation of the native enzyme resulted in modification of a methionine residue instead. These studies indicate that the two metal ions impose different structural and functional properties on the active site, leading to differing reactivities of specific amino acid residues. Replacement of zinc(II) in the methyltransferase enzyme MT2-A by cobalt(II) yields an enzyme with enhanced activity, where spectroscopy also indicates coordination by two thiolates and two histidines, supported by EXAFS analysis of the zinc coordination sphere.1210... 

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

Identification of carboxy-terminal amino acids was also attempted. Studies by Bergmann and his associates in the 1930s (see below) had characterized various peptidases with differing specificities. One of these was carboxypeptidase which required a free carboxy terminus adjacent to the peptide bond to be hydrolyzed. The specificity of the enzyme was limited but Lens in 1949 reported alanine to be at one end of insulin. Fromageot and his colleagues (1950) and Chibnall and Rees (1951) reduced the carboxy termini to B-aminoalcohols and showed glycine as well as alanine to be carboxy-terminal. Hydrazinolysis was also attempted the dry protein was treated with hydrazine at 100 °C for 6 h so that the carboxy-terminal amino acid was released as the free... 

In the case of carboxypeptidase B, Shaklai et al.(2lT> compared the relative contributions to the protein phosphorescence from tyrosine and tryptophan for the apoenzyme, the zinc-containing metalloenzyme in the absence of substrate, the metalloenzyme in the presence of the substrate iV-acetyl-L-arginine, and the metalloenzyme in the presence of the specific inhibitor L-arginine. The tyrosine tryptophan emission ratio of the metalloenzyme was about a factor of four smaller than that of the apoenzyme. Binding of either the substrate or the inhibitor led to an increase in the emission ratio to a value similar to that of the apoenzyme. The change in the tyrosine tryptophan phosphorescence ratio was attributed to an interaction between a tyrosine and the catalytically essential zinc. The emission ratio was also studied as a function of pH. The titration data are difficult to interpret, however, because a Tris buffer was used and the ionization of Tris is strongly temperature dependent. In general, the use of Tris buffers for phosphorescence studies should be avoided. 

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). 

To localize the rate of deuterium buildup to specific amides, the analyte protein is fragmented into a collection of peptides using combinations of endo- and exoproteases. Due to the low pH of the quench conditions in which the protein and peptide samples are maintained after deuterium labeling, acid-reactive proteases such as pepsin must be employed. Studies with combinations of acid-reactive en-doproteinases and carboxypeptidases have been employed to achieve greater sequence coverage and higher amide resolution [42, 45]. 

Drugs that have primary amino groups available for conjugation, for instance dopamine and doxorubicin, can in principle be coupled to LMWPs via oligopeptides. In contrast to the carboxypeptidases, the aminopeptidases appear to possess a broader specificity. To allow the release of terminal amino group-containing drugs in the acid environment of the lysosomes without the requirement of enzymes, an acid-sensitive spacer can be used. 

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. 

Zinc proteases carboxypeptidase A and thermolysin have been extensively studied in solution and in the crystal (for reviews, see Matthews, 1988 Christianson and Lipscomb, 1989). Both carboxypeptidase A and thermolysin hydrolyze the amide bond of polypeptide substrates, and each enzyme displays specificity toward substrates with large hydrophobic Pi side chains such as phenylalanine or leucine. The exopeptidase carboxypeptidase A has a molecular weight of about 35K and the structure of the native enzyme has been determined at 1.54 A resolution (Rees et ai, 1983). Residues in the active site which are important for catalysis are Glu-270, Arg-127, (liganded by His-69, His-196, and Glu-72 in bidentate fashion), and the zinc-bound water molecule (Fig. 30). 

T. L. Bullock, K. Breddam, S. J. Remington, Peptide aldehyde complexes with wheat serine carboxypeptidase II, implications for the catalytic mechanism and substrate specificity. /. Mol. Biol. 1996, 255, 714-725. 

Carboxypeptidase A"" (CPA, EC 3.4.17.1) is a proteolytic enzyme that cleaves C-terminal amino acid residues with hydrophobic side chains selectively. Several X-ray structures are available" The active site of CPA consists of a hydrophobic pocket (primary substrate recognition site) that is primarily responsible for the substrate specificity, a guanidinium moiety of Argl45 that forms hydrogen bonds to the carboxylate of the substrate, and Glu270, whose carboxylate plays a critical role, functioning either as a nucleophile to attack the scissUe carboxamide carbonyl carbon of the substrate or as a base to activate the zinc-bound water molecule, which in turn attacks the scissile peptide bond ". However, semiempirical calculations had shown that the direct attack of... 

Gas-liquid chromatography used for the determination of C-terminal amino acids and C-terminal amino acid sequences in nanomolar amounts of proteins was described in 1976 by Davy and Morris. Based on carboxypeptidase A digestion of the protein, the partially digested protein was removed and the amino acids released after known time intervals were analyzed by quantitative gas-liquid chromatography. Sequences deduced from the kinetics of release of specific amino acids are compared with the known C-terminal sequences of well-characterized proteins. Thus the amino acid sequences were determined. 

Zn+2, Mn+2, Fe+2, Cd+2, Co+2, and Ni+2, although diminished activity results (35, 53). Fundamentally the enolase and aconitase reactions are closely related, since the net result of both is the addition or subtraction of a molecule of water. In spite of this similarity, the metal ions associated with these two reactions are very different. It is one of the puzzling aspects of metalloenzyme chemistry that every enzyme has a different metal ion specificity. Each of these enzymes is associated in its natural state with a specific metal ion, which differs from enzyme to enzyme. It is possible to remove the naturally occurring metal from many of these enzymes and to reactivate them by the addition of other metals, as has been shown in the case of carboxypeptidase. The order in which the various metal ions fall in their ability to activate the different enzymes again varies from enzyme to enzyme. 

The enzymatic colorimetric format is followed by the Penzyme test. This test is a qualitative enzymatic assay for rapid detection of -lactam residues in milk (28-30). The detection principle of the Penzyme test is based on measurement of the degree of inactivation of the enzyme oo-carboxypeptidase is involved in the synthesis of the bacterial cell wall by -lactam antibiotics. These residues bind specifically with the enzyme and inactivate it, thus interfering with bacterial cell wall formation. 

There are five distinct families of zinc proteases, classified by the nature of the zinc binding site. These families, and their variously proposed mechanisms, have recently been reviewed in depth.143 The most studied member is the digestive enzyme bovine pancreatic carboxypeptidase A, which is a metalloenzyme containing one atom of zinc bound to its single polypeptide chain of 307 amino acids and Mr 34 472. It is an exopeptidase, which catalyzes the hydrolysis of C-terminal amino acids from polypeptide substrates, and is specific for the large hydrophobic amino acids such as phenylalanine. The closely related carboxypeptidase B catalyzes the hydrolysis of C-terminal lysine and arginine residues. The two en-... 

---