Carboxypeptidase characterization

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... 

Comparison of two analytical approaches, atomic force microscopy (AFM) and quartz crystal microbalance, for studying the binding of Con A to glycosylated carboxypeptidase, demonstrated that both could determine the quantitative parameters characterizing the interaction.65 Quantitative analyses of the interaction of Calreticulin (CRT), which is a soluble molecular chaperone of the endoplasmic reticulum, with various... 

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... 

Bayes, A., de la Vega, M. R., Vendrell, J., Aviles, F. X., Jongsma, M. A., Beekwilder, J. (2006). Response of the digestive system of Helicoverpa zea to ingestion of potato carboxypeptidase inhibitor and characterization of an uninhibited carboxypeptidase B. Insect Biochem. Mol. Biol., 36,654-664. 

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. 

Proteases of L. bulgaricus and L. helveticus contribute to the ripening of Swiss cheese (Langsrud and Reinbold 1973). Strains of thermo-duric lactobacilli are generally more proteolytic than S. thermophilus (Dyachenko et al. 1970). The proteinase activity of L. bulgaricus is optimal at pH 5.2-5.8 and is associated with the cell envelope (Argyle et al. 1976). Some strains of L. brevis (Dacre 1953) andL. lactis (Bottazzi 1962) are also proteolytic. Surface-bound aminopeptidase from L. lactis, characterized by Eggiman and Bachman (1980), is activated by cobalt and zinc ions and has optimum activity at pH 6.2-7.2. A surface-bound proteinase and carboxypeptidase are also present in L. lactis. 

The mechanism of action of carboxypeptidase has also been much studied recently by the techniques of cryoenzymology, which have allowed the identification and characterization of reaction intermediates.519,520 This has shown the presence of two intermediates during the hydrolysis of both peptides and esters. In conjunction with chemical evidence, this work demonstrates that there is no acyl intermediate in either peptide or ester hydrolysis, and that these two substrates form different metallo intermediates and are hydrolyzed through different mechanisms. 

In 1940 carbonic anhydrase was isolated from mammalian erythrocytes the protein was shown to contain 0.33% zinc. In 1955 carboxypeptidase became the second zinc enzyme to be reported. About 20 zinc metalloenzymes have since been studied in great detail, and about 60—70 await complete characterization. 

Shirley, A. M., and Chappie, C., 2003, Biochemical characterization of sinapoylglucose choline sinapoyltransferase, a serine carboxypeptidase-like protein that functions as an acyltransferase in plant secondary metabolism, J. Biol. Chem. 278 19870-19877. 

Carboxypeptidase A (EC 3.4.17.1) that cleaves carboxy-terminal aromatic or neutral amino acid from peptides and proteins has been extensively characterized [77], and... 

A saitoi acid carboxypeptidase is a glycoprotein that contains both N- and 0-1 inked sugar chains [81, 82], Glycosylation of the A. saitoi carboxypeptidase suggests the stability of this enzyme against proteinase and proteolysis. In this chapter, the characterization of a new N-linked high-mannose type oligosaccharide from A. saitoi acid carboxypeptidase is described. 

St. Leger, R. J., Bidochka, M. J., and Roberts, D. W. (1994b). Characterization of a novel carboxypeptidase produced by the entomopathogenic fungus Metarhizium anisopliae. Archives of Biochemistry and Biophysics, 314, 392-398. 

Blinkovsky, A. M., Byun, T., Brown, K. M., and Gohghtly, E. J. 1999. Purification, characterization, and heterologous expression in Fusarium venenatum of a novel serine carboxypeptidase from Aspergillus oryzae. Appl. Env. Microbiol., 65, 3298-3303. 

The metalloprotease carboxypeptidase catalyses the removal of C-terminal residues and is structurally related to the PM-located epidermal growth factor (EGF) receptor tyrosine kinase (see Chapter 8). Several plant carboxypeptidase inhibitor proteins have been characterized and the potato carboxypeptidase inhibitor (PCI) is also an EGF receptor antagonist (Table 13.4). 

Figure 6.1 Characterization of impaired prohormone processing in mice lacking functional active carboxypeptidase E (Cpe mice) by peptidomics.

In contrast to the aminopeptidases where the most extensive studies have been carried out on enzymes involved in hormone activation, the best characterized carboxypeptidase is from bovine pancreas having A-type activity. As indicated above, this enzyme probably serves exclusively to convert dietary protein into free amino acids. Accordingly the remaining discussion will deal with recent studies on its structure and mode of action. 

Figure 3 Cysteine protease and subtilisin-like protease pathways for proneuropeptide processing. Distinct cysteine protease and subtilisin-like protease pathways have been demonstrated for pro-neuropeptide processing. Recent studies have identified secretory vesicle cathepsin L as an important processing enzyme for the production of the endogenous enkephalin opioid peptide. Preference of cathepsin L to cleave at the NH2-terminal side of dibasic residue processing sites yields peptide intermediates with NH2-terminal residues, which are removed by Arg/Lys aminopeptidase. The well-established subtilisin-like protease pathway involves several prohormone convertases (PC). PC1/3 and PC2 have been characterized as neuroendocrine processing proteases. The PC enzymes preferentially cleave at the COOH-terminal side of dibasic processing sites, which results in peptide intermediates with basic residue extensions at their COOH-termini that are removed by carboxypeptidase E/H.

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