Active site of carboxypeptidase

Figure 7-3. Two-dimensional representation of a dipeptide substrate, glycyl-tyrosine, bound within the active site of carboxypeptidase A.

Nakagawa, S., and H. Umeyama. 1981. Molecular Orbital Study of the Effects of Ionic Amino Acid Residues on Proton Transfer Energetics in the Active Site of Carboxypeptidase A. Chem. Phys. Letters 81, 503-507. 

Peptidyl-dipeptidase A (angiotensin-I converting enzyme, ACE, EC 3.4.15.1) plays a pivotal role in the control of blood pressure [80]. It has been established that its active site contains an essential Zn-atom that functions like that of carboxypeptidase A [2], ACE is inhibited by peptides having a proline or aromatic amino acid at the C-terminal position. These observations as well as the similarities with the active site of carboxypeptidase A have allowed a rational design of effective inhibitors of ACE (e.g., captopril (3.4) and enalapril (3.5)) used in the treatment of hypertension [81]. 

Fig. 19. The carboxylate-histidine-zinc triad appears in the active site of carboxypeptidase A as Asp-142-His-69—and this interaction may contribute to zinc affinity in the metalloenzyme active site. Atomic coordinates were retrieved from the structure of the native enzyme (Rees et al, 1983) deposited in the Brookhaven Protein Data Bank.

Figure 12-16 Structure of the active site of carboxypeptidase A with a peptide substrate present. See Christianson and Lipscomb.409...

Stable compounds which resemble the transition-state structure of a substrate in an enzymatic reaction are expected to behave as potent reversible inhibitors (1 ). Based on the X-ray crystallographic structure of the active site of carboxypeptidase A (CPA) (2), a mechanism was proposed in which a water molecule adds directly to the scissile carbonyl group of the substrate to give the tetrahedral intermediate 1, which collapses to products (3). We proposed to mimic this tetrahedral intermediate, similar to the transition state, with the stable tetrahedral phosphonic acid derivatives 2,... 

Fig. 2.3.2. Carboxylate binding within the active site of Carboxypeptidase A.

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. 

Christianson, D. W. and Lipscomb, W. N. (1985) Binding of a possible transition state analog to the active site of Carboxypeptidase A, Proc. Natl. Acad. Sci. USA 82, 6840-6844. 

Figure 6 Amino acids in the active site of carboxypeptidase N. The dipeptide glycyl-L-tyrosine is docked into the active site. The active site zinc is coordinated to His 69, Glu 72, and His 196. Arg 145, Asn 144, and "tyr 248 provide specificity for substrates bearing a free terminal carboxylate. Gin 255 forms a hydrogen bond with the side chain of the carboxy-terminal arginine or lysine. Glu 270 promotes the nucleophilic attack of a water molecule at the scissile peptide bond.

Liu JQ, Wulff G (2004) Functional mimicry of the active site of carboxypeptidase A by a molecular imprinting strategy cooperativity of an amidinium and a copper ion in a transition-state imprinted cavity giving rise to high catalytic activity. J Am Chem Soc 126(24) 7452-7453... 

C-terminal amino acid while the converting enzyme detaches two. Furthermore, it was known that the active site of carboxypeptidase A comprises three important elements for the interaction with the substrate (Fig. 5.25) an electrophilic centre, establishing an ionic bond with a carboxylic function, a site capable of establishing a hydrogen bond with a peptidic C-terminal function, and an atom of zinc, solidly fixed on the enzyme and serving to form a coordinating bond with the carbonyl group of the penultimate (the scissile) peptidic function. 

Figure 1.4 Diagrammatic model of the active sites of carboxypeptidase-A and angiotensin converting enzyme and putative interaction modes with substrates and inhibitors (adapted from ref. 6).

Figure 6.6. Diagramatic model of the active site of carboxypeptidase A and hypothetical active site of ACE proposed by Cushman et al [83].

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 ... 

Several peptides isolated from the venom of the South American snake Bothrops jararaca are potent ACE inhibitors and were briefly used for the treatment of hypertension, but were soon superseded by surprisingly simple molecules with high inhibitory effect. At the Squibb Institute for Medical Research in Princeton, New Jersey, Miguel A. Ondetti (Plate 32) recognized that ACE is similar in its substrate specificity to the well studied protease car-boxypeptidase A. He designed, therefore, molecules that should fit into the active site of ACE (presumably similar to the active site of carboxypeptidase A) and form complexes with the enzyme. The dipeptide , 2-D-methyl-3-mercapto-propionyl-L-proline, captopril [7] strongly associated with the enzyme and... 

Several amino acid side chains at the active site of carboxypeptidase A participate in binding the substrate in the optimal position for reaction. Arg 145 forms two hydrogen bonds and Tyr 248 forms one hydrogen bond with the C-terminal carboxyl group of the substrate. (In this example, the C-terminal amino acid is phenylalanine.) The side chain of the C-terminal amino acid is positioned in a hydrophobic pocket, which is why carboxypeptidase A is not active if the C-terminal amino acid is arginine or lysine (Section 22.13). Apparently, the long, positively charged side chains of these amino acids (Table 22.2) cannot fit into the nonpolar pocket. 

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