Carboxypeptidase Catalytic mechanism

Uncovering of the three dimentional structure of catalytic groups at the active site of an enzyme allows to theorize the catalytic mechanism, and the theory accelerates the designing of model systems. Examples of such enzymes are zinc ion containing carboxypeptidase A 1-5) and carbonic anhydrase6-11. There are many other zinc enzymes with a variety of catalytic functions. For example, alcohol dehydrogenase is also a zinc enzyme and the subject of intensive model studies. However, the topics of this review will be confined to the model studies of the former hydrolytic metallo-enzymes. 

The evolutionary classification has a rational basis, since, to date, the catalytic mechanisms for most peptidases have been established, and the elucidation of their amino acid sequences is progressing rapidly. This classification has the major advantage of fitting well with the catalytic types, but allows no prediction about the types of reaction being catalyzed. For example, some families contain endo- and exopeptidases, e.g., SB-S8, SC-S9 and CA-Cl. Other families exhibit a single type of specificity, e.g., all families in clan MB are endopeptidases, family MC-M14 is almost exclusively composed of carboxypeptidases, and family MF-M17 is composed of aminopeptidases. Furthermore, the same enzyme specificity can sometimes be found in more than one family, e.g., D-Ala-D-Ala carboxypeptidases are found in four different families (SE-S11, SE-S12, SE-S13, and MD-M15). 

Carboxylic acids, solvent partition, 29 145-147 and dimerization, 29 145-147 Carboxypeptidase A, 22 409-421 active site, 22 418 catalytic mechanism, 22 416-421 chemical properties vs. Structure, 22 417-419... 

T. L. Bullock, K. Breddam, S. J. Remington, Peptide aldehyde complexes with wheat serine carboxypeptidase II, implications for the catalytic mechanism and substrate specificity. /. Mol. Biol. 1996, 255, 714-725. 

Cysteine proteases are so called because of a critical cysteine involved (together with an adjacent histidine) in the catalytic mechanism. Cysteine proteases include papain-related proteases, calpain-related proteases and the caspases. Papain-like cysteine proteases include the plant enzymes actinidin, aleurain, bromelain, caricain, chymopapain, ficin and papain and the lysosomal cathepsins B, C, H, K, L and S. Cathepsin C is multimeric (MW -200,000), but the other papain-related proteases are monomeric with MWs of about 20,000-35,000. While cathepsin C is a dipeptidyl aminopeptidase, the other enzymes are endopeptidases. Cathepsin B is an endopeptidase and a dipeptidyl carboxypeptidase. Cathepsin H is an endopeptidase and an aminopeptidase. In higher animals, cathepsin B generates peptides from antigens for presentation to T cells by the major histocompatibility... 

It is well established that the same three-dimensional scaffolding in proteins often carries constellations of amino acids with diverse enzymatic functions. A classic example is the large family of a/jS, or TIM, barrel enzymes (Farber and Petsko, 1990 Lesk et ai, 1989). It appears that lipases are no exception to date five other hydrolases with similar overall tertiary folds have been identified. They are AChE from Torpedo calif arnica (Sussman et al., 1991) dienelactone hydrolase, a thiol hydrolase, from Pseudomonas sp. B13 (Pathak and Ollis, 1990 Pathak et al, 1991) haloalkane dehalogenase, with a hitherto unknown catalytic mechanism, from Xanthobacter autotrophicus (Franken et al, 1991) wheat serine carboxypeptidase II (Liao et al, 1992) and a cutinase from Fusa-rium solani (Martinez et al, 1992). Table I gives some selected physical and crystallographic data for these proteins. They all share a similar overall topology, described by Ollis et al (1992) as the a/jS hydrolase... 

Figure 8. Proposed catalytic mechanism for carboxypeptidase A where Glu270 acts as the nucleophile a) nucleophilic attack by carboxylate oxygen of Glu270 on the carbonyl carbon of the substrate b) forming a tetrahedral intermediate c) the intermediate breaks down to give an anhydride species d) hydrolysis of the acylenzyme leads to product formation and regnerates the free enzyme...

Aminopeptidases are counterparts to carboxypeptidases, removing N-terminal amino acids. However, unlike the carboxypeptidases, they contain dinuclear zinc sites. They fall into two groups, the first of which includes the leucine aminopeptidase from bovine lens, while the second includes the leucine aminopeptidases AAP from Aeromonas proteolytica and SAP from Streptomyces griseus (Figure 12.15). The mechanism of the AAP enzyme has been well studied, and may well represent a general catalytic mechanism for peptide hydrolysis by metal-lopeptidases with a cocatalytic active site.ki. 

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). 

Once the hydrolysis has been performed, the cleaved amino acid still interacts with Arg-145 and with the hydrophobic pocket, whereas the amino group interacts with Glu-270. The carboxylate group of the new terminal amino acid interacts with zinc. This picture, which is a reasonable subsequent step in the catalytic mechanism, finds support from the interaction of L- and D-phenylala-nine with carboxypeptidase. >3i-i34,i38... 

Johansen and Vallee 176) and Quiocho et al. 177) have carried out experiments both in solution and in the crystal phase with a chemically modified, Tyr-248, enzyme derivative to examine the relationship between the conformational state of the residue and the catalytic mechanism. Derivatization of Tyr-248 with dicizotized arsanihc acid to form arsanilazo-Tyr-248 carboxypeptidase A... 

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). 

Figure 12.9 Proposed catalytic mechanism for carboxypeptidase A. The C-terminal residue, R represent a bulky, hydrophobic side chain. Carboxypeptidase (EC3.3.4.17.-) promotes the polarization of the scissile carbonyl group by hydrogen bonding to Argl27, the activation of water molecule by Zn and its deprotonation by Glu270. The zinc-hydroxide ion attack on the carbonyl carbon forms the tetrahedral oxyanion transition state. The formation of products requires protonation of the amino leaving group presumably by Glu270...

Figure 3.4 Catalytic mechanism of yeast carboxypeptidase Y mediated peptide hyrolysis and a model for the acyltransferase activity of SMT.

Enzymes that cleave polypeptide chains within the chain at any susceptible point away from the N and C termini. They are subdivided according to the catalytic mechanism or preference for certain amino acids into serine, aspartic, and cysteine. These enzymes attack proteins producing mainly smaller peptides. Endopeptidases and carboxypeptidases act synergistically. 

Similar reaction mechanisms, involving general base and metal ion catalysis, in conjunction with an OH nucleophilic attack, have been proposed for thermolysin (Ref. 12) and carboxypeptidase A (Refs. 12 and 13). Both these enzymes use Zn2+ as their catalytic metal and they also have additional positively charged active site residues (His 231 in thermolysin and... 

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. 

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