Carboxypeptidase A

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Carboxypeptidase A in Bos taurus

1 Introduction
2 Structure
- 2.1 Active Site
3 Important Tyr248 Residue
4 Mechanism of Action
5 Catalytic and Inhibitory Zinc Binding
6 Other Inhibitors

Introduction

Figure 1: Catalytic and inhibitory Zn²⁺ ions in the active site of 1CPX. The catalytic and inhibitory Zn²⁺ ions are shown in cyan and red, respectively. PDB code: 1CPX.

is a metalloexopeptidase whose biological function is to cleave the C-terminal amino acid residue from polypeptide substrates.^[1] 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.^[2] 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.^[1] 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 (shown in cyan), while the binding of a second Zn²⁺ inhibits the hydrolysis reaction mechanism (shown in red) (Figure 1).^[1] An example of a crystal structure for active CPA (one zinc bound) has been deposited in the Protein Data Bank (PDB) database as 3CPA. An inhibited CPA (two zincs bound) has been deposited under the label 1CPX.

Structure

Bovine CPA exists as a single unit with C1 symmetry in the pancreatic physiological environment. According to structural information deposited in the PDB database, the single polypeptide chain of CPA contains a mixture of , of which there are a total of 11 helices (one 3₁₀, eight 3.6₁₃) and ten β-strands. The helices are shown in magenta, and the β-strands are displayed in yellow. A disulfide bond connects the residues Cys138 and Cys161. The disulfide bond can be seen in yellow in the .

Six different biologically active forms of the CPA monomeric unit exist. 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 α-form (residues Ala1-Asn307) or the β-form (residues Ser3-Asn307). Chymotrypsin cleavage generates the γ-form (residues Asn8-Asn307). The α-form essentially is the protein without any additional residue cleavages. The Ala and Arg residues, shown in red and white respectively, are cleaved in the β-form. In addition to the red and white residues, the residues displayed in yellow are cleaved to give the γ-form. The of CPA arise from genetic variation in residues located at three separate positions of the polypeptide chain. The differences include the following: Ile/Val179, Ala/Glu228, and Val/Leu305.^[1] 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 (colored orange) whose opening is located on the surface of the protein. When no polypeptide substrate is bound in the active site, the pocket is open. However, the pocket is (see section titled "Important Tyr248 Residue") when a substrate or inhibitor molecule binds.^[2] In this view, the Tyr248 is shown in green. Kinetics experiments have indicated that the binding region of the active site is actually capable of extending over five amino acids of the substrate.^[2] 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 Hydrophobic Binding Pocket

The (spacefill view, subsite in green) 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 (pseudo-mesh view, subsite in green). 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 . 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.^[1]

S1 Subsite

In the same way that the S1' subsite is involved in anchoring the polypeptide substrate in place, the (spacefill view, subsite in cyan) contains several residues that help hold the substrate in the active site, but the S1 subsite also contains the residues that are involved in the catalytic chemical mechanism. In general, the residues of the (pseudo-mesh view, subsite in cyan) 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 () aid in the recognition of the C-terminal residue of a polypeptide substrate.^[1] 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 (see Figure 3 in the section titled "Mechanism of Action"). helps stabilize the substrate in the active site by engaging in ion-dipole interactions with the carbonyl oxygen of the penultimate substrate residue (Figure 3). Three residues () 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 3).^[2] deprotonates this water molecule and acts as a base catalyst in the hydrolysis mechanism (Figure 3). , along with the positively charged Zn²⁺ ion, help stabilize the negatively charged intermediate generated in the addition-elimination step of the hydrolysis reaction (Figure 3).^[2]

Putting It All Together

For a 3D manipulative applet displaying the essential amino acid residues and their interactions with a bound ligand, . The hydrophobic binding pocket (shown in yellow) can be seen interacting with a phenylalanine side chain of the C-terminal amino acid. The Zn²⁺ ion is held in place by the three side chains mentioned previously (shown in green). The Tyr248 side chain is explicitly shown in cyan and is "capping" the pocket since a substrate is bound. The base catalyst, Glu270, is explicitly shown in orange. The other side chains in the darker blue color are part of the stabilizing side chains of the S1 subsite. Manipulate the scene manually, and zoom in and out to see the interactions between CPA's side chains and the bound substrate. This crystal structure with the bound ligand, which is actually a phosphonate, has been deposited in the Protein Data Bank (PDB) with code 6CPA.

Important Tyr248 Residue

CPA from B. taurus has been co-crystallized with two Zn²⁺ ions (Figure 1). This structure has been deposited in the PDB database under the label 1CPX, which is a β-form of CPA. 1CPX has an interesting distinction from some other crystallized CPA proteins in that its crystallographic data has revealed a different conformation of the Tyr248 residue.

Figure 2: Important Tyr248 residue of 1CPX is pointing toward the hydrophobic binding pocket.

^[1] Previous literature has suggested that the conserved Tyrosine residue among CPA proteins has been involved in an induced fit mechanism because Tyr248 typically has been found toward solution when a substrate is not bound in the active site.^[1] However, crystallographic data for 1CPX shows Tyr248 (Figure 2) without a polypeptide substrate bound. Therefore, this conflicts with the previously proposed induced fit mechanism for CPA proteins and suggests that Tyr248 is liganded to the catalytic Zn²⁺ ion in 1CPX.^[1] Moreover, this data supports that this is the native conformation of Tyr248 in solution because none of the residues in the protein’s active site interact in crystallographic packing.^[1] Not only does Tyr248 point toward the active site, but in doing so, Tyr248 (shown in blue) with its interactions with Ile247 and a hydrogen bond.^[1] This point of view is able to clearly show how Tyr248 is thought to be a ligand with the catalytic Zn²⁺ ion.

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.^[2] 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.^[3]

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 3) is as follows:

Three S1 subsite residues (Asn144, Arg145, and Tyr248) and the hydrophobic S1' subsite recognize the C-terminus of the polypeptide substrate.
After aiding in the recognition of the substrate, Tyr248 "caps" the binding pocket.
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.
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.
The peptide bond is cleaved through an addition-elimination step.
The Glu270 base catalyst is regenerated through a final proton transfer with the nitrogen atom of the former C-terminal peptide bond.
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.

Figure 3: Hydrolysis of C-terminal polypeptide substrate residue by CPA using the promoted water pathway. Residues of the S1 subsite stabilize the negatively charged intermediate once the water molecule complexed with the Zn²⁺ ion is deprotonated by the base catalyst, Glu270, and attacks the carbonyl. This figure is derived from Figure 10 in "Carboxypeptidase A" by Christianson and Lipscomb (Acc. Chem. Res., 1989).^[2]

Catalytic and Inhibitory Zinc Binding

As previously stated, CPA from B. taurus has the ability to bind two Zn²⁺ ions in its active site. The binding of only one Zn²⁺ ion is catalytic, while the binding of a second is inhibitory. These Zn²⁺ ions are connected to each other via a hydroxy-bridge (Figure 4) with a distance of 3.48 Å.^[1]

Figure 4: Hydroxy-bridge between catalytic and inhibitory zinc ions. The catalytic Zn2+ ion (shown in orange on the right) is bridged to the inhibitory Zn2+ ion (shown in orange on the left) by a OH- (shown in red).

Figure 4: Hydroxy-bridge between catalytic and inhibitory zinc ions. The catalytic Zn²⁺ ion (shown in orange on the right) is bridged to the inhibitory Zn²⁺ ion (shown in orange on the left) by a OH^- (shown in red).

In the CPA structure containing only the catalytic Zn²⁺ ion , a water molecule complexed to the zinc is able to be deprotonated by Glu270 to allow for normal initiation of hydrolysis (Figure 3). However, when is also present (1CPX), it occupies the physical space that would normally be occupied by the water molecule. Thus, the inhibitory Zn²⁺ ion interacts with the carboxylate group of Glu270. The Glu270 now simply stabilizes the second Zn²⁺ and is unable to perform its usual base catalyst role while the catalytic Zn²⁺ ion (shown in green) is still being stabilized in place by His69, Glu72, and His196 (shown in orange).

Other Inhibitors

In addition to the inhibitory Zn²⁺ ion, CPA can be inhibited by other ions and molecules. Some of these inhibitors include, but are not limited to, other metal ions and anions,^[4] phosphonates,^[5] cysteine, sulfides, and cyanide, the chelating agent 1,10-pentathroline,^[6] Ochratoxin A,^[7] and Latexin.^[8]

Detailed studies of anions have indicated that the nature of anion inhibition is partly competitive in the binding site.^[1] In particular, the sulfate anion (SO₄^2-) has been of interest to researchers. In a crystallized structure of carboxypeptidase T (PDB code: 1ORD), a SO₄^2- anion is found occupying a portion of a region that corresponds to the Arg127, Asn144, Arg145, and Tyr248 residues in the S1 subsite of carboxypeptidase A.^[1] In this case, it is understood that the SO₄^2- anion prevents the recognition of the carboxylate group at the C-terminus of the polypeptide substrate.

References

↑ ^1.00 ^1.01 ^1.02 ^1.03 ^1.04 ^1.05 ^1.06 ^1.07 ^1.08 ^1.09 ^1.10 ^1.11 ^1.12 ^1.13 Bukrinsky JT, Bjerrum MJ, Kadziola A. 1998. Native carboxypeptidase A in a new crystal environment reveals a different conformation of the important tyrosine 248. Biochemistry. 37(47):16555-16564. DOI: 10.1021/bi981678i
↑ ^2.0 ^2.1 ^2.2 ^2.3 ^2.4 ^2.5 ^2.6 Christianson DW, Lipscomb WN. 1989. Carboxypeptidase A. Acc. Chem. Res. 22:62-69.
↑ Suh J, Cho W, Chung S. 1985. Carboxypeptidase A-catalyzed hydrolysis of α-(acylamino)cinnamoyl derivatives of L-β-phenyllactate and L-phenylalaninate: evidence for acyl-enzyme intermediates. J. Am. Chem. Soc. 107:4530-4535. DOI: 10.1021/ja00301a025
↑ Geoghegan, KF, Galdes, A, Martinelli, RA, Holmquist, B, Auld, DS, Vallee, BL. 1983. Cryospectroscopy of intermediates in the mechanism of carboxypeptidase A. Biochem. 22(9):2255-2262. DOI: 10.1021/bi00278a031
↑ Kaplan, AP, Bartlett, PA. 1991. Synthesis and evaluation of an inhibitor of carboxypeptidase A with a Ki value in the femtomolar range. Biochem. 30(33):8165-8170. PMID: 1868091
↑ Worthington, K., Worthington, V. 1993. Worthington Enzyme Manual: Enzymes and Related Biochemicals. Freehold (NJ): Worthington Biochemical Corporation; [2011; accessed March 28, 2017]. Carboxypeptidase A. http://www.worthington-biochem.com/COA/
↑ Pitout, MJ, Nel, W. 1969. The inhibitory effect of ochratoxin a on bovine carboxypeptidase a in vitro. Biochem. Pharma. 18(8):1837-1843. DOI: 0.1016/0006-2952(69)90279-2
↑ Normant, E, Martres, MP, Schwartz, JC, Gros, C. 1995. Purification, cDNA cloning, functional expression, and characterization of a 26-kDa endogenous mammalian carboxypeptidase inhibitor. Proc. Natl. Acad. Sci. 92(26):12225-12229. PMCID: PMC40329

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Michael Melbardis, Douglas Schnell, Thomas Baldwin, Geoffrey C. Hoops, Michal Harel

Retrieved from "http://52.214.119.220/wiki/index.php/Carboxypeptidase_A"

@@ Line 13: / Line 13: @@
 ===Active Site===
-The active site of bovine pancreatic CPA is embedded within a <scene name='69/694222/3cpadeeppocket/1'>deep pocket</scene> (colored orange) whose opening is located on the surface of the protein.  When no polypeptide substrate is bound in the active site, the pocket is open. However, the pocket is "capped" by a tyrosine residue (Tyr248) (see section titled "Important Tyr248 Residue") when a substrate or [http://en.wikipedia.org/wiki/Enzyme_inhibitor inhibitor] molecule binds.<ref name="CPA2" />  Kinetics experiments have indicated that the binding region of the active site is actually capable of extending over five amino acids of the substrate.<ref name="CPA2" />  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.
+The active site of bovine pancreatic CPA is embedded within a <scene name='69/694222/3cpadeeppocket/1'>deep pocket</scene> (colored orange) whose opening is located on the surface of the protein.  When no polypeptide substrate is bound in the active site, the pocket is open. However, the pocket is <scene name='69/694222/3cpadeeppocket2/1'>"capped" by a tyrosine residue (Tyr248)</scene> (see section titled "Important Tyr248 Residue") when a substrate or [http://en.wikipedia.org/wiki/Enzyme_inhibitor inhibitor] molecule binds.<ref name="CPA2" />  In this view, the  Tyr248 is shown in green.  Kinetics experiments have indicated that the binding region of the active site is actually capable of extending over five amino acids of the substrate.<ref name="CPA2" />  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 Hydrophobic Binding Pocket=====
@@ Line 26: / Line 26: @@
 ==Important Tyr248 Residue==
 CPA from ''B. taurus'' has been  co-crystallized with two Zn<sup>2+</sup> ions (Figure 1).  This structure has been deposited in the PDB database under the label [http://www.rcsb.org/pdb/explore/explore.do?structureId=1cpx 1CPX], which is a β-form of CPA.  1CPX has an interesting distinction from some other crystallized CPA proteins in that its crystallographic data has revealed a different conformation of the Tyr248 residue.[[Image:1CPX-Tyr248.png|thumb|Figure 2: Important Tyr248 residue of 1CPX is pointing toward the hydrophobic binding pocket.]]<ref name="CPA1">Bukrinsky JT, Bjerrum MJ, Kadziola A. 1998. Native carboxypeptidase A in a new crystal environment reveals a different conformation of the important tyrosine 248. ''Biochemistry''. 37(47):16555-16564. [http://pubs.acs.org/doi/abs/10.1021/bi981678i DOI: 10.1021/bi981678i]</ref>  Previous literature has suggested that the conserved Tyrosine residue among CPA proteins has been involved in an [http://en.wikipedia.org/wiki/Enzyme_catalysis#Induced_fit induced fit mechanism] because Tyr248 typically has been found <scene name='69/694222/Tyr248_5cpa/1'>pointing outward</scene> toward solution when a substrate is not bound in the active site.<ref name="CPA1">Bukrinsky JT, Bjerrum MJ, Kadziola A. 1998. Native carboxypeptidase A in a new crystal environment reveals a different conformation of the important tyrosine 248. ''Biochemistry''. 37(47):16555-16564. [http://pubs.acs.org/doi/abs/10.1021/bi981678i DOI: 10.1021/bi981678i]</ref>  However, crystallographic data for 1CPX shows Tyr248 <scene name='69/694222/Tyr248_1cpx/1'>pointing toward the active site</scene> (Figure 2) without a polypeptide substrate bound.  Therefore, this conflicts with the previously proposed induced fit mechanism for CPA proteins and suggests that Tyr248 is liganded to the catalytic Zn<sup>2+</sup> ion in 1CPX.<ref name="CPA1">Bukrinsky JT, Bjerrum MJ, Kadziola A. 1998. Native carboxypeptidase A in a new crystal environment reveals a different conformation of the important tyrosine 248. ''Biochemistry''. 37(47):16555-16564. [http://pubs.acs.org/doi/abs/10.1021/bi981678i DOI: 10.1021/bi981678i]</ref>  Moreover, this data supports that this is the native conformation of Tyr248 in solution because none of the residues in the protein’s active site interact in crystallographic packing.<ref name="CPA1">Bukrinsky JT, Bjerrum MJ, Kadziola A. 1998. Native carboxypeptidase A in a new crystal environment reveals a different conformation of the important tyrosine 248. ''Biochemistry''. 37(47):16555-16564. [http://pubs.acs.org/doi/abs/10.1021/bi981678i DOI: 10.1021/bi981678i]</ref>  Not only does Tyr248 point toward the active site, but in doing so, Tyr248 <scene name='69/694222/Tyr248_pointing_toward/2'>caps the hydrophobic binding pocket</scene> (shown in blue) with its interactions with Ile247 and a hydrogen bond.<ref name="CPA1">Bukrinsky JT, Bjerrum MJ, Kadziola A. 1998. Native carboxypeptidase A in a new crystal environment reveals a different conformation of the important tyrosine 248. ''Biochemistry''. 37(47):16555-16564. [http://pubs.acs.org/doi/abs/10.1021/bi981678i DOI: 10.1021/bi981678i]</ref>  This point of view is able to clearly show how Tyr248 is thought to be a ligand with the catalytic Zn<sup>2+</sup> ion.
-For a space-fill view of Tyr248 capping the binding pocket, <scene name='69/694222/3cpadeeppocket2/1'>click here</scene>.  In this view, the Tyr248 is shown in green, while the binding pocket is displayed in orange.
 == Mechanism of Action ==