Carboxypeptidase A and the Role of Zinc

Enzymes that hydrolyze amide and ester bonds may be divided into three classes (1) those requiring a thiol group for activity, such as papain, ficin, and other plant enzymes (2) those inhibited by diisopropylphosphorofluo-ridate (DFP), such as a-chymotrypin, trypsin, subtilisin, cholinesterase, and thrombin (3) those that require a metal ion for activity. This last class includes dipeptidases, and exopeptidases such as carboxypeptidase and leucine aminopeptidase. The metal ion is involved in the stabilization of the tetrahedral intermediate (refer to Section 4.4.1). 

Another important metalloenzyme is carbonic anhydrase which catalyzes the conversion of carbon dioxide into bicarbonate ion. It is a zinc-dependent 

X-Ray studies of carbonic anhydrase show that the active center is composed of three imidazole ligands which have distorted tetrahedral coordination to the Zn(II) ion. Molecular models suggested that a similar geometry could be attained with a tris(imidazolyl) methane derivation. For this reason, R. Breslow s team (218) in 1978 synthesized tris[4(5)imidazolyl] carbinol (4-TIC) and tris(2-imidazolyl) carbinol (2-TIC) as models for the zinc binding site of carbonic anhydrase and alkaline phosphatase. Similarly, bis[4(5)- 

Comparison between the different models clearly showed that 4-TIC is a tridentate ligand capable of using all three imidazole rings for coordination to Zn(II), Co(II), or Ni(II). 

In this section we will orient the rest of the discussion on carboxypeptidase A. This is a zinc-dependent enzyme which catalyzes the hydrolysis of C-terminal amino acid residues of peptides and proteins and the hydrolysis of the corresponding esters. This exopeptidase has a molecular weight of 34,600 and a specificity for aromatic amino acid in the L-configuration. 

---

The collapse of the proteolytic tetrahedral intermediate of the promoted-water pathway requires a proton donor in order to facilitate the departure of the leaving amino group. Rees and Lipscomb (1982) considered Glu-270, but favored Tyr-248 for this role, but Monzingo and Matthews (1984) fully elaborated on a role for Glu-270 of carboxypeptidase A and Glu-143 of thermolysin as intermediate proton donors. This proposal for carboxypeptidase A is corroborated by the near-normal activity observed for the Tyr-248- Phe mutant of rat carboxypeptidase A (Garden et al, 1985 Hilvert et al, 1986) and is reflected in the mechanistic scheme of Fig. 31 (Christianson and Lipscomb, 1989). Mock (1975) considered Glu-270 a proton donor in the carboxypeptidase A mechanism, but his mechanism does not favor a Glu-270/zinc-promoted water molecule as the hydrolytic nucleophile. Schepartz and Breslow (1987) observed that Glu-270 may mediate an additional proton transfer in the generation of the Pi product carboxylate. 

Zinc usually binds to proteins via residues of cysteine and histidine. Sometimes zinc is bound to residues of glutamate or aspartate. The zinc ion sometimes plays a catalytic role and sornetimes a structural role. In the latter case, it helps maintain the three-dimensional structure or conformation of the protein. For example carboxypeptidase A contains twr> atoms of zinc. One is required for catalytic activity and is bound to cysteine and histidine. The other, which plays a structural role, is bound only hr cysteine. Cytoplasmic supeioxide dismutase is a dimer, ft contains one atom of Cu and one of Zn + per subunit. The zinc is bound via three residues of histidine and one residue of aspartate. It is buried deep within the enzyme and serves a structural role. The copper atom is bound via four residues of histidine. It resides dose to the surface of the protein and participates in the chemistry of catalysis. 

The molecular details of the action of metalloenzymes have begun to be elucidated in the past few years (42). Crystal structures for bovine carboxypeptidase A (43), thermolysin (44), and horse liver alcohol dehydrogenase (45) are now available, and chemical and kinetic studies have defined the role of zinc in substrate binding and catalysis. In fact, many of the significant features elucidating the mode of action of enzymes in general have been defined at the hands of zinc metalloenzymes. 

Carboxypeptidase A is one of the most intensely investigated zinc metalloenzymes. The enzyme as isolated contains 1 g-atom of zinc per protein molecular weight of 34,600. Removal of the metal atom either by dialysis at low pH or by treatment with chelating agents gives a totally inactive apoenzyme (46). Activity can be restored by readdition of zinc or one of a number of other di-valent metal ions (47). Through a combined use of chemical modification and transient state kinetic studies, it has been possible to determine the role of zinc in the catalysis of ester and peptide hydrolysis by this enzyme. 

Although zinc, cadmium, and mercury are not members of the so-called main-group elements, their behavior is very similar because of their having complete d orbitals that are not normally used in bonding. By having the filled s orbital outside the closed d shell, they resemble the group IIA elements. Zinc is an essential trace element that plays a role in the function of carboxypeptidase A and carbonic anhydrase enzymes. The first of these enzymes is a catalyst for the hydrolysis of proteins, whereas the second is a catalyst for the equilibrium involving carbon dioxide and carbonate,... 

One of the most important metals with regard to its role in enzyme chemistry is zinc. There are several significant enzymes that contain the metal, among which are carboxypeptidase A and B, alkaline phosphatase, alcohol dehydrogenase, aldolase, and carbonic anhydrase. Although most of these enzymes are involved in catalyzing biochemical reactions, carbonic anhydrase is involved in a process that is inorganic in nature. That reaction can be shown as... 

Zinc is a microelement essential for proper functioning of the human body. The level of daily demand for zinc was established as 13 to 16 mg (Ziemlahski, 2001). Zinc plays a role in protein and carbohydrate metabolism and is a component of over 60 metaloenzymes, including alkaline phosphatase, pancreatic carboxypeptidases A and B, alcoholic and lactic dehydrogenases, carbonate anhydrase, and proteases. It also forms bonds with nucleic acids -which is very important for their functioning (Prasad, 1983 Valee and Falchuk, 1993). 

We have already seen a number of models for the zinc(II) containing enzymes such as carbonic anhydrase in Section 11.3.2. Zinc is an essential component in biochemistry, and forms part of the active site of more then 100 enzymes, of which hydrolases (such as alkaline phosphatase and carboxypeptidase A), transferases (e.g. DNA and RNA polymerase), oxidoreductases (e.g. alcohol dehydrogenase and superoxide dismutase) and lysases (carbonic anhydrase) are the most common. In addition, the non-enzyme zinc finger proteins have an important regulatory function. In many of these systems, the non-redox-active Zn2+ ion is present as a Fewis acidic centre at which substrates are coordinated, polarised and hence activated. Other roles of zinc include acting as a template and playing a structural or regulatory role. 

Generally speaking, the role of the enzyme consists of the selective and specific attraction of substrate and the highly efficient catalysis. Every enzyme has its own characteristic feature for example, the specificity in the binding and a charge-relay action in the catalysis in a-chymotrypsin, the contribution of the imidazole moiety as an electron donor to the electrophilicity of zinc ion in carboxypeptidase, the change in the spin state and the reactivity of the transition metal ion by the coordination of the imidazole in the hemochrome. These typical characteristic features are the result of the cooperative actions of the constituents. 

One of the most important discoveries concerning the biological role of zinc occurred in 1940 when Keilin and Mann showed that zinc is an essential compound of erythrocyte carbonic anhydrase, an enzyme cata-lytically involved in the transport of CO2 in blood (6). Following the 70-year interval between the initial recognition of a metabolic zinc deficiency and the characterization of the first zinc metalloenzyme, there was a period of about 15 years before the second zinc enzyme was identified. In 1955, Vallee and Neurath reported that carboxypeptidase A from bovine pancreas contained 1 g-atom Zn per mol of protein and was essential to the function of the enzyme (7). The presence of zinc in carbonic anhydrase and carboxypeptidase A indicated that a primary role of zinc would be to function in zinc metalloenzymes (62). However, it seemed unhkely that disrupting the activity of carboxypeptidase A or carbonic anhydrase would have profound eflFects on growth. 

The crystal structures of several complexes of the metallo enzyme, carboxypeptidase A (CPA)(EC 3.4.17.1), have been examined in considerable detail. The structure of the complex with glycyl tryosine (Gly-Tyr) as been refined to 2.0 A resolution and reveals inter alia interactions between the amide carbonyl oxygen and the catalytically essential zinc, and between the amide nitrogen and the hydroxyl of tryosine-248 (Tyr-248)(Fig. 11). The proposed mechanisms for hydrolysis of peptide and ester bonds by CPA have relied heavily on these crystal structures, but a clear distinction between the possible roles of glutamate-270 (Glu-270) in nucleophilic attack either by general base catalysis (Fig. 11 A) or by covalent any hydride formation (Fig. IIB) remains a major unresolved problem. Indeed, it is not yet certain whether esters and amides are hydrolyzed by CPA via identical mechanisms. 

Based on his study on model compounds, Breslow suggested a second mechanism for peptide hydrolysis by carboxypeptidase A which does not involve the formation of an acyl-enzyme intermediate (221,222). Essentially, in the hydrolysis of a peptide bond there is participation of a zinc ion, a carboxylate ion, and a tyrosine hydroxyl group. The Zn(II) ion still plays the role of a Lewis acid to coordinate the carbonyl oxygen but the carboxylate group rather acts as a general base. The argument is based on the fact that... 

The matrix metalloprotease (MMP) family of zinc hydrolases are thought to play important roles in extracellular tissue remodeling in angiogenesis and other normal physiological processes, in some inflammatory processes and in metastatic processes in cancer. Like the zinc carboxypeptidases, the MMPs also utilize a zinc-coordinated water molecule to initiate attack on the scissile amide bond of protein substrates. These enzymes are synthesized by the ribosome in a latent form composed of a catalytic domain and an N-terminal extension, referred to as the prodomain the latent, or inactive form of the enzyme is referred to as a zymogen or... 

---