CPA carboxypeptidase

RNS, ribonuclease LZM, lysozyme SNS, staphylococcal nuclease LZ4, T4 lysozyme PAP, papain TLS, thermolysin, TRX, thioiedoxin FLN, flavodoxin ADH, alcohol dehydrogenase coenzyme domain AKN, adenyl kinase MDG, malate dehydrogenase TIM, triosephosphate isomerase SUB, subtilisin CPA, carboxypeptidase LDH, lactate dehydrogenase PGK, phosphoglycerate kinase GPD, glyceraldehyde 3-phosphate dehydrogenase, HKN, hexokinase. 

GOUP-TF, chicken ovalbumin upstream promoter transcription factor COX, cyclooxygenase COX-1, cyclooxygenase 1 COX-2, cyclooxygenase 2, inducible cyclooxyge n ase CPA, carboxypeptidase... 

Fig. 30. Important active-site residues of carboxypeptidase A (CPA) and thermolysin (TLN) and a general scheme for the active sites of related zinc proteases.

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... 

The crystal structure of the complex formed between carboxypeptidase Aa (abbreviated CPA) and glycyltyrosine (Gly-Tyr) has been refined to 2.0 A by Lipscomb et at. (444, 445) and it reveals (Fig. 90) interactions between the amide carbonyl oxygen and the catalytically essential Zn, and between the amide nitrogen and the hydroxyl of tyrosine-248 (Tyr-248). Scott et al. (443) synthesized both the [13C]amido (90% enriched) and amido[l3C, 15N]amido (90% and 99% enriched, respectively) isotopomers of Gly-Tyr. They then proceeded to probe the hydrolysis by a series of l3C and l5N high-resolution solid-state NMR spectra. 

Carboxypeptidase A (3.4.17.1) Carboxypeptidase A3 MC-CPA, RMC-CP Zinc EDTA, chitosan-EDTA conjugates [64], poly(acrylate) derivatives [49], polycarbophil-cysteine [67]... 

By the use of a model system, Kimura et al. [17] tried to mimic the function of the two mechanistically most typical zinc(II) enzymes. Carbonic anhydrase (CA, EC 4.2.1.1) catalyses the reversible hydration of carbon dioxide to bicarbonate ion and its zinc(II) active site is bound to three histidine residues and a water molecule. Carboxypeptidase A (CPA, EC 3.4.17.1) catalyses the hydrolysis of the hydrophobic C-terminal amino acids from polypeptides, and its active-site zinc(II) is bound to two histidine residues, a glutamine residue and a water molecule which is hydrogen bound to a glutamine residue (Scheme 19). 

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,... 

The combination of Lewis add and nucleophile activations in 12-14 requires four-membered ring transition states. Interestingly, the mechanism for carboxypeptidase A (CPA) catalyzed hydrolysis of peptides also appears to involve joint Lewis add and nucleophile activations that lead to the formation of a four-membered ring transition state. Christianson and Lipscomb [57] have determined the crystal structure (15) of a ketone bound to CPA. Surprisingly, the ketone is in its hydrated form with both oxygens of the gem-diol bound to the active-site zinc of CPA (Figure 6.12). 

The vertebrate pancreatic carboxypeptidases are one such family of digestive enzymes. Within the family, two broad substrate preferences are known. Enzymes with carboxypeptidase A (CPA) activity cleave hydro-phobic and aromatic residues from the carboxyl terminus of peptides and proteins, whereas those with carboxypeptidase B (CPB) activity prefer substrates with arginine and lysine residues at the C terminus.3 Several duplicates within the family have CPA-like activity but display a range of substrate preferences.5,6... 

Carboxypeptidase (CPA) Crystalline carboxypeptidase isolated by John Northrop (USA, Nobel Prize, Chemistry Chemistry, 1946, pure enzyme viral protein isolation) 13.5D... 

Solanum Carboxypeptidase inhibitor (= PCI) Solanum tuberosum (potato) (Solanaceae) [tuber] CPA [2 nMJ [ EGF-R antagonist]... 

The kinetics of action of carboxypeptidase are very complex. The pH-rate profile for CPA-catalyzed hydrolysis of peptides is bell shaped, with apparent pK values of about 6.5 and 7.5. The former pK value could be attributed to Glu-270. The pH dependency of the kinetics of the CPA-catalyzed enolization of the ketonic substrate (R)-2-benzyl-3-(p-methoxybenzoyl)pro-pionic acid leads to the establishment (with minimum complications from this relatively simple reaction) of a pK value of 6.03 for the Co" and Zn" CPA, which is probably due to Glu-270. The binding of the substrate to both Zn" and Co" CPA appears to depend on an enzyme-bound group with pXa = 7.56 and 8.29 respectively, and on a group with pXa>9. These are attributed to the ionization of Tyr-248 and the bound water molecule. 

Gray and coworkers " have prepared copper(II) carboxypeptidase A. Cu"CPA, and compared its spectrum, that of the enzyme with inhibitor present, and those of several other copperdl) complexes with nitrogen and oxygen ligating atoms. Some of these data together with the geometry about the copper ion are ... 

The most studied member of zinc proteases is the digestive enzyme bovine pancreatic carboxypeptidase A (CPA) which is a metalloenzyme containing one atom of zinc bound to its single polypeptide side chain of 307 amino acids with a molecular weight of 34 kD. It is an exopeptidase, which catalyses the hydrolysis of C-terminal amino... 

The second example in this chapter is the carboxypeptidase A (CPA) [42, 43]. CPA is an exo-peptidase which can hydrolyze the C terminal amino acid from the peptide or ester substrates, whose X-ray structures have been reported for its native form [44, 45] or enzyme-inhibitor complex [46-51]. In addition, an X-ray stmc-mre of enzyme complexed with the proteolysis product was also reported [52]. No matter accumulation of experimental data, its reaction mechanisms still remain incompletely understood [53]. In particular, two major mechanisms, promoted-water pathway and nucleophilic pathway (traditionally it was named as anhydride pathway), using a peptide as the model substrate are depicted in Fig. 9.4. The nucleophilic pathway envisages an acyl-enzyme (AE) intermediate resulting from direct... 

Fig. 9.4 Putative mechanisms for the hydrolysis of peptides catalyzed by CPA [67]. Reprinted with permission from (Wu S, Zhang C, Xu D, Guo H (2011) pH-dependent reactivity for gly-cyl-L-tyrosinein carboxypeptidase-A-catalyzed hydrolysis, JPhys Chem B 115(34)10360-10367). Copyright (2011) American Chemical Society...

Circular dichroism (CD) has played an important role in our studies on the modification of enzymes and hormones with Co(III). The objective of these studies has been to incorporate selectively substitution inert metal ions at specifically modified sites in proteins as probes of biological function. Significant information concerning the catalytic mechanism of carboxypeptidase A (CPA) (1) has been obtained from a site specific modification of tyrosine 248 with Co(III) (2). The method developed for CPA has been extended to other enzymes and hormones in order to devg op an improved method for incorporating stable radioisotopes t Co) into proteins. The substitution-inertness of Co(III) provides the necessary stability in these derivatives (3). 

In their classic study they were able to modify specifically tyrosine 248 in the zinc metalloenzyme, carboxypeptidase A, to give the azotyrosine derivative, arsanilazotyrosine 248 carboxypeptidase A (AA-CPA-Zn), shown in Figure 3A. The native Zn is shown explicitly in order to differentiate it from externally incorporated Co as will be discussed. They found that at intermediate pH s, where the enzyme exhibits maximal activity, the azotyrosine is chelated to the intrinsically bound active site zinc. A distinct red color is associated with zinc chelation in contrast to the yellow and orange colors of the enzyme due to the presence of the free azophenol (low pH) and azophenolate (high pH), respectively (7). 

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