Introduction

is a metalloexopeptidase whose biological function is to cleave the C-terminal amino acid residue from polypeptide substrates. Specifically, CPA is one member of a large group of Zn²⁺ metalloenzymes that carries out the hydrolysis of C-terminal polypeptide residues through the deprotonation of a water molecule that is coordinated to the Zn²⁺ ion in the enzyme's active site. CPA consists of a single polypeptide chain that contains 307 amino acids. Produced in the pancreas, CPA itself must first be modified by trypsin and chymotrypsin in order to achieve an active form that serves its biological function. Although different biologically active forms of CPA are found across different species, including humans, much research has investigated bovine pancreatic zinc carboxypeptidase A. X-ray crystallography has demonstrated that bovine CPA has the ability to bind two Zn²⁺ ions in its active site, in which the binding of one Zn²⁺ is catalytic, while the binding of a second Zn²⁺ inhibits the hydrolysis reaction mechanism.

Structure

Bovine CPA exists as a monomeric unit with C1 symmetry in the pancreatic physiological environment. The single polypeptide chain contains a mixture of α-helices and β-sheets, of which there are a total of 11 helices (one 3₁₀, eight 3.6₁₃) and ten β-strands. These secondary structural characteristics can be observed in Figure 1.

Six different biological active forms of the monomeric CPA unit exist. Three of these active forms are produced following the cleavage of amino acid residue segments from the initial zymogen, or proenzyme, by trypsin and chymotrypsin, which are also found in the pancreas. Cleavage by trypsin generates either the alpha-form (residues Ala1-Asn307) or the beta-form (residues Ser3-Asn307). Chymotrypsin cleavage generates the gamma-form (residues Ser3-Asn307). The other three active forms of CPA arise from genetic variation in residues located in three separate positions of the polypeptide chain. The differences include the following: Ile/Val179, Ala/Glu228, and Val/Leu305. Each of the six biologically active monomeric units carry out the same function of hydrolyzing the C-terminal peptide bond of a polypeptide substrate.

Active Site

The active site of bovine pancreatic CPA is embedded within a deep pocket whose opening is located on the surface of the protein. When no polypeptide substrate is bound in the active site, the pocket is open, but the pocket is "capped" by a tyrosine residue (Tyr248) when a substrate or inhibitor molecule binds. Kinetics experiments have indicated that the binding region of the active site is actually capable of extending over five amino acids of the substrate. The active site contains two separate subsites, labeled S1' and S1, which each contain several pertinent residues that serve important roles during the catalyzed hydrolysis reaction.

S1' Subsite

The S1' subsite contains multiple hydrophobic residues that interact by van der Walls forces with C-terminal hydrophobic side chains of polypeptide substrates. For this reason, the S1' subsite is referred to as the hydrophobic binding pocket. It should be of note that the S1' subsite, despite being named as a hydrophobic pocket, is not another pocket in addition to the deep pocket active site. Rather, it is simply a particular region of the deep pocket. Specifically, the hydrophobic pocket includes the residues Ile243, Ile247, Ala250, Gly252, Gly253, Ser254, and Ile255. The hydrophobic nature of the S1' subsite assists in establishing some degree of specificity for CPA. Because the hydrophobic pocket anchors the polypeptide substrate in the active site, the larger and more hydrophobic the side chain of the C-terminal substrate residue, the stronger the van der Walls interactions between the subsite and the substrate. Therefore, the stability of substrate binding is increased when residues like phenylalanine are present at the C-terminus. Essentially, the S1' subsite serves as a recognition site for the C-terminal side chain of the substrate.

S1 Subsite

In the same way that the S1' subsite is involved in anchoring the polypeptide substrate in place, the S1 subsite contains several residues that help hold the polypeptide substrate in place, but the S1 subsite also contains the residues that are involved in the catalytic chemical mechanism. In general, the residues of the S1 subsite have polar or charged side chains that either allow for hydrogen bonding to stabilize negatively charged intermediates of the hydrolysis reaction or position particular atoms appropriately to allow for chemistry to occur. Three residues (Asn144, Arg145, and Tyr248) aid in the recognition of the C-terminal residue of a polypeptide substrate. The Asn144 and Tyr248 residues each engage in hydrogen bonding and ion-dipole interactions with the carboxyl group at the C-terminus, while Arg145 provides additional stability by participating in ion-ion interactions with the carboxyl group (Figure ***). Arg71 helps stabilize the substrate in the active site by engaging in ion-dipole interactions with the carbonyl oxygen of the penultimate substrate residue (Figure ***). Three residues (His196, Glu72, and His69) are liganded to a catalytic Zn2+ ion that is complexed to a water molecule positioned one bond distance away from the C-terminal peptide bond carbonyl carbon (Figure ***). Glu270 deprotonates this water molecule and acts as a base catalyst in the hydrolysis mechanism (see Figure *** in the section titled "***"). Arg127, along with the positively charged Zn2+ ion, help stabilize the negatively charged intermediate generated in the addition-elimination step of the hydrolysis reaction (Figure ***).

Mechanism of Action

Two chemical mechanisms have been proposed for the hydrolysis reaction catalyzed by CPA. One mechanism, referred to as the nucleophilic pathway, involves a covalent acyl enzyme intermediate (an anhydride intermediate) containing Glu270, the active site base. Although there is some chemical and kinetic support for the nucleophilic pathway, the evidence is mixed and ambiguous. In one set of experiments conducted by Suh and his colleagues in 1985, accumulation of an intermediate (assumed to be the acyl enzyme) was obtained; however, the intermediate was isolated without confirmation by trapping experiments. Therefore, the conclusions of the study only provide marginal evidence for the mixed anhydride intermediate.

The second mechanism, which has been coined as the promoted water pathway, is better supported by chemical and structural data. The mechanism of the reaction (Figure ***) is as follows:

1. Three S1 subsite residues (Asn144, Arg145, and Tyr248) and the hydrophobic S1' subsite recognize the C-terminus of the polypeptide substrate.
2. After aiding in the recognition of the substrate, Tyr248 "caps" the binding pocket.
3. The catalytic Zn²⁺ ion and Arg127 residue engage in ion-dipole interactions with the oxygen atom of the carbonyl group of the C-terminal peptide bond, further polarizing the carbon to oxygen double bond.
4. A water molecule, which has been deprotonated by Glu270 (base catalyst) and is being held in place one bond distance away from the partially positive carbon of the C-terminal carbonyl, acts as a nucleophile and attacks this carbon to generate a tetrahedral intermediate stabilized by both the Zn²⁺ ion and surrounding positive charges of S1 subsite residues.
5. The peptide bond is cleaved through an addition-elimination step.
6. The Glu270 base catalyst is regenerated through a final proton transfer with the nitrogen atom of the former C-terminal peptide bond.
7. Product release is facilitated, in part, by unfavorable electrostatic interactions between the regenerated Glu270 base catalyst and the deprotonated carboxylic acid at the new C-terminus.

Catalytic and Inhibitory Zinc Binding

Other Ligands

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