Carboxyl groups Carboxypeptidase

The peptidases are divided into endopeptidases and exopeptidases. The endopeptidases (formerly proteinases) are able to attack all the peptide bonds in a molecule, even those which are some distance from terminal groups, whilst the exopeptidases (formerly peptidases) can only hydrolyse peptide bonds at the ends of the chain. Among the exopeptidases, some remove terminal residues having a free carboxyl group (carboxypeptidases) whilst others remove those where the amino group is free (aminopepti-... 

U. H. Mortensen, S. J. Remington, K. Breddam, Site-Directed Mutagenesis on (Serine) Carboxypeptidase Y. A Hydrogen Bond Network Stabilizes the Transition State by Interaction with the C-Terminal Carboxylate Group of the Substrate , Biochemistry 1994, 33, 508-513. 

There are three fundamental questions concerning the mechanism of action of carboxypeptidase (1) How do metal ions affect ester and amide hydrolysis (2) Under what conditions will a neighbouring carboxyl group participate in ester and amide hydrolysis emd what is the mechanism of such participation (3) How will a metal ion affect... 

An important difference between thermolysin and carboxypeptidase leads to the major uncertainty in the mechanism of carboxypeptidase. This difference is that the catalytic carboxylate of carboxypeptidase is far more sterically accessible. The crucial question is whether or not the carboxypeptidase-catalyzed hydrolysis of peptides proceeds via general-base catalysis, as in equation 16.26, or via nucleophilic catalysis, as in 16.27. Early kinetic work concentrated on establishing the participation of the various groups in catalysis. 

The COOH-terminal amino acid of a peptide or protein may be analyzed by either chemical or enzymatic methods. The chemical methods are similar to the procedures for NH2-terminal analysis. COOH-terminal amino acids are identified by hydrazinolysis or are reduced to amino alcohols by lithium borohydride. The modified amino acids are released by acid hydrolysis and identified by chromatography. Both of these chemical methods are difficult, and clear-cut results are not readily obtained. The method of choice is peptide hydrolysis catalyzed by carboxypeptidases A and B. These two enzymes catalyze the hydrolysis of amide bonds at the COOH-terminal end of a peptide (Equation E2.3), since carboxypeptidase action requires the presence of a free a-carboxyl group in the substrate. 

There are simple reagents that react selectively with the carboxyl terminus of a peptide, but they have not proved as generally useful for analysis of the C-terminal amino acids as has the enzyme carboxypeptidase A. This enzyme catalyzes the hydrolysis of the peptide bond connecting the amino acid with the terminal carboxyl groups to the rest of the peptide. Thus the amino acids at the carboxyl end will be removed one by one through the action of the enzyme. Provided that appropriate corrections are made for different rates of hydrolysis of peptide bonds for different amino acids at the carboxyl end of the peptide, the sequence of up to five or six amino acids in the peptide can be deduced from the order of their release by carboxypeptidase. Thus a sequence such as peptide-Ser-Leu-Tyr could be established by observing that carboxypeptidase releases amino acids from the peptide in the order Tyr, Leu, Ser ... 

In the acylation step a nucleophilic group on one of the amino-acid side chains at the active site behaves as the nucleophile. As we have seen in Section 25-9B, the nucleophile of carboxypeptidase is the free carboxyl group of glutamic acid 270. In several other enzymes (chymotrypsin, subtilisin, trypsin, elastase, thrombin, acetylcholinesterase), it is the hydroxyl group of a serine residue ... 

Although many of these general features are correct, these mechanistic conclusions are based upon the assumption that the properties of the enzyme in solution are conserved on crystallization. It appears from Raman studies on arsanilazotyrosine-248 carboxypeptidase that the enzyme exists in solution in a number of different forms.509 It is not certain that the form which crystallizes out is the kinetically active species. Indeed there is evidence that the kinetics of carboxypeptidase in solution differ from those of the enzyme crystals, with the crystalline enzyme being 1000-fold less active with some substrates.510 The interaction of Gly-L-tyr with Mn carboxypeptidase A, studied by 1H NMR techniques, does not involve coordination of the carboxyl group of the substrate, and may well represent a different conformation from the one studied by crystallographic techniques.511 Several conformational forms of the Cd11 carboxypeptidase A have been found in solution, while the enzyme exists in a different form in the crystal state.312... 

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... 

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. 

Comparison of the deduced sequence of A. saitoi carboxypeptidase with other known serine carboxypeptidase sequences shows that they share a low degree of similarity 32% with wheat carboxypeptidase II, 32.3% with malt carboxypeptidase II and 26.2% with yeast carboxypeptidase Y (Figure 19) [88], However, all of the sequences conserve the catalytic domains (indicated by boxes II to IV in Figure 19) and the domain (box I in the Figure 19) which contains the amino acid residues recognizing the C-terminal carboxylate group of peptide substrates. There are also present in the sequence ten potential sites for N-linked glycosylation. 

Zinc proteinases contain a tightly-bound active site Zn2 + ion and a carboxylate group in the two zinc proteinases whose X-ray crystal structures are known, carboxypeptidase A and thermolysin, these are Glu-270 and Glu-143 (Lipscomb, 1983). One thus has at least the following three possibilities for the initial catalytic event ... 

In carboxypeptidase A [52, 53], the active-site Zn(n) ion plays essential catalytic roles and the guanidinium of Arg-145 recognizes the carboxylate anion of the substrates, thus making the enzyme an exopeptidase. Important features of carboxypeptidase A reproduced by 11 include the essential catalytic action of a metal ion and participation of a guanidinium group in substrate recognition, so that this polymer biocatalyst hydrolyzes unactivated amides, and exhibits selectivity toward amide bonds adjacent to carboxylate groups in the substrate. 

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