Carboxypeptidases cleavage sites

The peptide maps obtained on in situ reactions are often incomplete due to compound-specific suppression (Nielsen and Roepstorff, 1989). In situ reactions are especially valuable to confirm a tentative identification of a peptide based on the determined molecular mass. In such cases confirmation of one or more predicted cleavage sites is sufficient. In situ carboxypeptidase digestions are also less effeaive than digestions carried out in solution. Frequently only the C-terminal residue is identified or the sequence of a few residues can be determined (Klarskov et al., 1989) however, this information is obtained without further sample use and often it is sufficient for confirmation of a peptide identity or a valuable supplement to N-terminal sequencing by Edman degradation. 

Many structural biology and biochemistry studies rely on the precise identification of the N-/C-terminal sequence of a protein (e.g., signal peptide identification, determining the cleavage site and specificity of a novel protease, etc.). Digestion of proteins in arrays of carboxypeptidase Y at different concentrations with MALDl-MS-based readout can be employed to derive C-terminal sequence information for proteins. The amino-terminus of a protein can be derived by wet chemistries similar to Edman degradation coupled to MS-readout. This method of protein ladder sequencing, reported first by Kent and... 

As mentioned earlier, by far the largest number of zinc enzymes are involved in hydrolytic reactions, frequently associated with peptide bond cleavage. Carboxypeptidases and ther-molysins are, respectively, exopeptidases, which remove amino acids from the carboxyl terminus of proteins, and endopeptidases, which cleave peptide bonds in the interior of a polypeptide chain. However, they both have almost identical active sites (Figure 12.4) with two His and one Glu ligands to the Zn2+. It appears that the Glu residue can be bound in a mono- or bi-dentate manner. The two classes of enzymes are expected to follow similar reaction mechanisms. 

The binding of glycyl-L-tyrosine in the active site pocket of carboxypeptidase A is illustrated in Fig. 15. Tyrosine-248 and glutamic acid-270 are believed to participate in the catalytic reaction and represent the acidic and basic groups, respectively, involved in the bell-shaped pK-rate profile. In the bond-cleavage reaction, the carboxyl group of Glu-270 may act by a nucleophilic attack on the carbonyl group while Tyr-248... 

In models for carboxypeptidase A we showed the intracomplex catalyzed hydrolysis of an ester by a metal ion and a carboxylate ion [106], which are the catalytic groups of carboxypeptidase A. Some mechanistic proposals for the action of carboxypeptidase involve an anhydride intermediate that then hydrolyzes to the product and the regenerated enzyme. Although we later found convincing evidence that the enzyme does not use the anhydride mechanism in cleaving peptides [96-99], it may well use such a mechanism with esters. In a mimic of part of this mechanism we showed [107], but see also Ref. 108, that we could achieve very rapid hydrolysis of an anhydride by bound Zn2+, which is the metal ion in the enzyme. In another model, a carboxylate ion and a phenolic hydroxyl group, which are in the enzyme active site, could cooperatively catalyze the cleavage of an amide by the anhydride mechanism [109]. 

During last decades the domains C-2 symmetry (the dyad rotation symmetry) of low-B palindrome was established in many enzymes (chymotrypsin, trypsin, aspartyl proteinases, HIV-1 protease, carboxypeptidase A, phospholipase A-2 ribonuclease, etc.) (Lumry, 2002 and references therein). It is proposed that the pair domain closure causes constrain of pretransition state complex that activates cleavage or formation of chemical bonds. Thus control of strong bonds by the cooperation of many matrix or knots bonds takes place. As an example, in the active site of carboxypeptidase A the zinc ion is attached to one of the catalytic domains by histidine 69 and glutamine 72 and connected by hystidine 196 to the second domain. Similar structures were found in the chymotrypsin and pepsin active sites where protons are driven under compression of the domains closure. 

Zinc enzymes catalyze the hydrolysis of amide bonds using a variety of active site structural motifs.77 An extensively studied enzyme of this class is carboxypeptidase A, which contains a mononuclear zinc center (Fig. 13) within the enzyme active site and catalyzes the hydrolysis of a C-terminal amino acid.78,79 The mechanism of amide cleavage in carboxypeptidase A has been studied extensively.77,78 In one proposed mechanistic pathway for this enzyme (Scheme 13), termed the zinc hydroxide mechanism , the zinc center activates a water molecule for deprotonation and also assists in polarization of the substrate carbonyl moiety, thus making it more susceptible to nucleophilic attack. The zinc center also provides transition state stabilization through charge neutralization. 

Carboxypeptidase A (CPA) is a pancreatic metaUoenz5Tne which catalyses the cleavage of a peptide link in a polypeptide chain. The site of cleavage is specific in two ways it occurs at the C-terminal amino acid (equation 28.22), and it exhibits a high selectivity for substrates in which the C-terminal amino acid contains a large aliphatic or Ph substituent. The latter arises from the presence, near to the active site, of a hydro-phobic pocket in the protein which is compatible with the accommodation of, for example, a Ph group (see below). 

Carboxypeptidase A (EC 3.4.17.1) is a pancreatic digestive enzyme that consists of a single polypeptide chain of 307 amino acids with a total of 36,000. It catalyzes the cleavage of amino acid residues from C-termini of polypeptides. Importantly, for its mechanism of action, it contains one Zn +in its active site. The amino acid side chains that form its active site and the catalytic sequence are shown in Fig. 5-27. 

Carboxypeptidase is a proteolytic enzyme that cleaves the C-terminal amino acid residue from a polypeptide. The amino acid arginine is found in the active site of carboxypeptidase and is responsible for holding the C-terminal end of the polypeptide in place so the cleavage of the peptide bond can occur. What type of interaction occurs between the C-terminal end of the polypeptide and the arginine side chain ... 

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