Mechanisms carboxypeptidase-catalyzed hydrolysis

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. 

M. Perryman, D. Knell, and R. Roberts. Carboxypeptidase-catalyzed hydrolysis of C-terminal lysine mechanism for in vivo production of multiple forms of creatine kinase in plasma. Clin. Chem. 30 662-664 (1984). 

THE MECHANISM The mechanism shown outlines the major stages in carboxypeptidase-catalyzed hydrolysis of a peptide in which the C-terminal amino acid is phenylalanine. Proton transfers accompany stages 2 and 3 but are not shown. Only the major interactions of the substrate with the carboxypeptidase side chains are shown although others may also be involved. 

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]. 

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). 

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...

Carboxypeptidase A has esterase activity as well as peptidase activity. In other words, the compound can hydrolyze ester bonds as well as peptide bonds. When carboxypeptidase A hydrolyzes ester bonds, Glu 270 acts as a nucleophilic catalyst instead of a general-base catalyst. Propose a mechanism for the carboxypeptidase A-catalyzed hydrolysis of an ester bond. 

R. Breslow, D. E. McClure, R. S. Brown, and J. Eisenach (1975), Very fast zinc catalyzed hydrolysis of an anhydride. A model for the rate and mechanism of carboxypeptidase A catalysis. /. Amer. Chem. Soc. 97,194-195. 

Knowing how the protein chain is folded is a key ingredient m understanding the mechanism by which an enzyme catalyzes a reaction Take carboxypeptidase A for exam pie This enzyme catalyzes the hydrolysis of the peptide bond at the C terminus It is... 

FIGURE 27 19 Proposed mechanism of hydrolysis of a peptide catalyzed by carboxypeptidase A The peptide is bound at the active site by an ionic bond between its C terminal ammo acid and the positively charged side chain of arginine 145 Coordination of Zn to oxygen makes the carbon of the carbonyl group more positive and increases the rate of nucleophilic attack by water... 

There are five distinct families of zinc proteases, classified by the nature of the zinc binding site. These families, and their variously proposed mechanisms, have recently been reviewed in depth.143 The most studied member is the digestive enzyme bovine pancreatic carboxypeptidase A, which is a metalloenzyme containing one atom of zinc bound to its single polypeptide chain of 307 amino acids and Mr 34 472. It is an exopeptidase, which catalyzes the hydrolysis of C-terminal amino acids from polypeptide substrates, and is specific for the large hydrophobic amino acids such as phenylalanine. The closely related carboxypeptidase B catalyzes the hydrolysis of C-terminal lysine and arginine residues. The two en-... 

Mechanisms similar to the one described for carboxypeptidase appear to operate in the hydrolysis of amide and ester bonds catalyzed by a number of pro-... 

P-CD derivatives have also been used to catalyze the hydrolysis of esters, mimicking the mechanism employed by carboxypeptidases. Carboxypepti-dase A is a metalloenzyme that is much more effective than chymotrypsin in hydrolyzing amide bonds. Zinc in carboxypeptidase A has a typical... 

This complex has been shown to be an excellent structural and functional model for the zinc hydrolytic enzymes, particularly carbonic anhydrase but also carboxypeptidase and the zinc phosphate esterases (24-26). The same complex also catalyzes the hydration of acetaldehyde and hydrolysis of carboxylic esters. These reactions appear to progress via a mechanism similar to that proposed for carbonic anhydrase. The rates are slower for [Zn([12]aneN3)OH] than for the enzyme but an order of magnitude faster than for existing model systems such as [(NH3)5Co(OH)]2+ (26). 

Proteins are hydrolyzed very slowly with storage in water at neutral pH. However, addition of proteases can increase the rate of hydrolysis about 10 billion times over the spontaneous rate. The chymotrypsin mechanism depicted in Figure 2.53 is shared by trypsin and elastase. These three proteases are members of a family called the serine proteases (named after Ser 195), Carboxypeptidase Aand pepsin catalyze peptide hydrolysis by different mechanisms and are not part of this family. 

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. 

In support of the mechanism of R, accumulation of intermediates during the carboxypeptidase A-catalyzed ester hydrolysis has been reported (57, 58), and several lines of evidence were consistent with the nucleophilic attack of Glu(270) leading to the anhydride intermediate (59, 60). Other pieces of evidence have been presented for the esterase action of carboxypeptidase A in support of the anhydride mechanism, such as resonance Raman spectroscopic measurements or trapping of the anhydride intermediate with a radioactive... 

The DhiA enzyme functions as a monomer ( 35 kDa) and is composed of two domains a main domain and a cap domain (Figure 2(a)). The main domain consists of a mostly parallel eight-stranded /3-sheet connected by ct-helices on both sides of the sheet. The cap domain is composed of five ct-helices with intervening loops. The active site is an occluded hydrophobic cavity located at the interface of the two domains. The overall fold of the main domain is the hallmark of the o //3-hydrolase fold superfamily of enzymes, to which lipases, esterases, carboxypeptidases, and acetylcholinesterases also belong. These superfamily members catalyze the hydrolysis of ester and amide bonds via a two-step nucleophilic substitution mechanism similar to that of serine proteases. 

Most exopeptidases are metalloproteases (exceptions e.g. D-amino acid aminopep-tidase, Salmonella methionine aminopeptidase). Aminopeptidases catalyze the hydrolysis of amino acid residues from the N-terminus of peptide substrates with broad substrate specificity. However, carboxypeptidases hydrolyze C-terminal amino acids with varied substrate specificity. Carboxypeptidase A, which prefers large hydrophobic side chain for the C-terminal residue of peptide substrates, has been extensively investigated (Christianson and Lipcomb, 1989) and its catalytic mechanism is illustrated in Figure 12.9. An analogous mechanism has been proposed for the requiring aminopeptidases (Taylor, 1993). 

Therefore, a key argument in regard to the proposed enzymatic mechanism of carboxypeptidase A is whether the carboxylate group of Glu-270 is steri-cally capable of participating efficiently in a nucleophilic reaction. In fact, such evidence has now been obtained by spectral characterization in the subzero temperature range (—60°C) study of a covalent acyl-enzyme intermediate obtained in the hydrolysis of the specific substrate 0-(trans-p-chlo-rocinnamoyl)-L-jS-phenyllactate by carboxypeptidase A (224). Furthermore, the results indicate that deacylation of the mixed anhydride intermediate is catalyzed by a Zn-bound hydroxide group. 

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