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== 1CPX - Carboxypeptidase A from Bovine Pancreas ==
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=Trehalose-''O''-mycolyltransferase Ag85C=
 
==Introduction==
==Introduction==
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Antigen 85C is one of three homologous protein components of the Ag85 complex in the cell wall of ''M. tuberculosis''. This serine esterase enzyme catalyzes the transfer of mycolyl groups, characteristic components of the cell wall of mycobacteria. Several three dimensional structures of Ag85C have been solved, including the wild type enzyme as well as active site variants due to site-directed mutagenesis and covalent modification.
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<scene name='69/694222/3cpaoverview/1'>Carboxypeptidase A (peptidyl-L-amino acid hydrolase, EC 3.4.17.1, often abbreviated CPA)</scene> is a metallo[http://en.wikipedia.org/wiki/exopeptidase exopeptidase] that hydrolytically cleaves peptides at the [http://en.wikipedia.org/wiki/C-terminus C-terminal] peptide bond containing hydrophobic side chains. <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> Specifically, CPA is a Zn<sup>2+</sup> [http://en.wikipedia.org/wiki/Metalloprotein#Metalloenzymes metalloenzyme] that carries out the hydrolysis of C-terminal polypeptide residues through the [http://en.wikipedia.org/wiki/Deprotonation deprotonation] of a water molecule that is coordinated to the Zn<sup>2+</sup> ion in the enzyme's [http://en.wikipedia.org/wiki/Active_site active site].<ref name="CPA2">Christianson DW, Lipscomb WN. 1989. Carboxypeptidase A. ''Acc. Chem. Res.'' 22:62-69.</ref> From bovine pancreas, 1CPX has been crystallized in a new environment alongside two Zn<sup>2+</sup> ions, one catalytic, the other inhibitory. The inhibitory Zn<sup>2+</sup> ion in this more recent crystal structure not only prevents 1CPX from undergoing its [http://en.wikipedia.org/wiki/hydrolysis hydrolysis] mechanism; but also, this crystallographic data reveals a different conformation of the Tyr248 (GREEN LINK) residue suggesting alternative mechanistic behavior. <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> 1CPX consists of a single polypeptide chain that contains 307 amino acids. CPA proteins must first be activated by either [http://en.wikipedia.org/wiki/Trypsin trypsin] or [http://en.wikipedia.org/wiki/Chymotrypsin chymotrypsin] in order to achieve an active form that serves its biological function.<ref name="CPA1" /> This crystallographic data presents 1CPX activated by trypsin in its β-form and orthorhombic crystal form.
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<StructureSection load='1dqz' size='400' side='right' caption='Antigen 85C in ''Mycobacterium Tuberculosis''' scene=''>
 
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==Biological Role==
 
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===Cell Wall of Mycobacteria===
 
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The unusually thick and waxy cell wall of [http://en.wikipedia.org/wiki/Mycobacterium mycobacteria] is primarily composed of [http://en.wikipedia.org/wiki/Peptidoglycan peptidoglycans], [http://en.wikipedia.org/wiki/Arabinogalactan arbinogalactans], and [http://en.wikipedia.org/wiki/Mycolic_acid mycolic acids]. The mycolic acids, which have very long carbon chains and exhibit extreme hydrophobicity, are responsible for forming the outermost layer of the cell wall, thus creating a hydrophobic envelope surrounding the mycobacterium. Much of the mycolic acid content of the cell wall is in the form of esters (trehalose-6-monomycolate, TMM) and bis-esters (trehalose-6,6'-dimycolate, TDM, [http://en.wikipedia.org/wiki/Cord_factor cord factor]) of [http://en.wikipedia.org/wiki/Trehalose trehalose].
 
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===Enzymatic Activity===
 
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The antigen 85 (Ag85) complex in [http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis ''Mycobacterium tuberculosis''] is composed of three homologous proteins: Ag85A, Ag85B, and Ag85C, encoded by the ''fbpA'', ''fbpB'', and ''fbpC'' genes, respectively. Each of these Ag85 components (A, B, and C) is an acyltransferase enzyme (EC 2.3.1.122) that can catalyze mycolyl transfer from TMM to another alcoholic substrate, including TMM itself (Figure 1).
 
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[[Image:MycolylTransferReaction.jpg|700 px|center|thumb|'''Figure 1:''' A mycolyl transfer reaction catalyzed by Ag85C]]
 
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===Clinical Relevance===
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This is about 1CPX. This is a carboxypeptidase from bovine pancreas with two Zinc binding sites.This is about 1CPX. This is a carboxypeptidase from bovine pancreas with two Zinc binding sites.This is about 1CPX. This is a carboxypeptidase from bovine pancreas with two Zinc binding sites.This is about 1CPX. This is a carboxypeptidase from bovine pancreas with two Zinc binding sites.This is about 1CPX.
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The hydrophobic envelope created by these inclusion of mycolic acids in the cell walls of mycobacteria results in a barrier against small hydrophilic molecules, such as Tuberculosis antibiotics. Thus, inhibition of Ag85C offers potential for cell wall disruption and subsequent antibiotic targeting for normally drug-resistant ''Mycobacteria tuberculosis''. <ref>PMID: 10200974</ref>
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<scene name='75/752350/Tyr_248_zoom/1'>active site</scene>
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==Structure of Wild Type Enzyme==
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This is a carboxypeptidase from bovine pancreas with two Zinc binding sites.
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[[Image:Substrate Binding 2D Surface.jpg|200 px|left|thumb|'''Figure 2A:''' Octylthioglucoside (green), a substrate analog, shown in the binding pocket of Ag85C (tan, from [[1va5]])]]
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[[Image:Octylthioglucoside.jpg|200 px|left|thumb|'''Figure 2B:''' 1-''O''-Octyl-β-D-thioglucoside]]
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Wild type Ag85C, in the absence of any specific ligands, was originally crystallized as a <scene name='69/694219/Dimer/1'>homodimer</scene>.<ref name="Ronning2000">PMID: 10655617</ref> The <scene name='69/694220/Secondary_structures/2'>secondary structure</scene> (shown in the monomeric form) is 38% α-helical (pink) and 16% β-sheet (yellow). The confrontation of a central β-sheet bordered by α–helices comprises an [http://en.wikipedia.org/wiki/Alpha/beta_hydrolase_fold α/β hydrolase fold] in Ag85C, a supersecondary structure that is highly conserved across serine hydrolases. The active site of Ag85C is composed of two adjoined binding pockets; there is carbohydrate binding pocket for the trehalose substrate, and there is a fatty acid binding pocket for the mycolic acid. As a result, trehalose monomycolate can effectively bind to the Ag85C active site. This active site is shown in Figure 2, in which Ag85C is shown with a bound substrate mimic, octylthioglucoside<ref name="Ronning2004">PMID: 15192106</ref> (Figure 3).
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<Structure load='1CPX' size='350' frame='true' align='left' caption='This is a caption about 1CPX' scene='75/752350/Tyr_248_zoom/1' />
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===Catalytic Triad===
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[[Image:1CPX Cartoon.png|200 px|right|thumb|Figure Legend]]
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[[Image:Substrate_Catalytic_Triad.jpg|300 px|right|thumb|'''Figure 3:''' Relation of the catalytic triad to the octylthioglucoside analog in [http://www.rcsb.org/pdb/explore/explore.do?structureId=1VA5 Ag85C]]]
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Mutagenesis studies<ref name="Favrot2014"> PMID:25028518 </ref> have confirmed the the catalytic function of Ag85C is dependent upon a Glu-His-Ser <scene name='69/694220/Catalytic_triad/4'>catalytic triad</scene>, similar to that of [http://en.wikipedia.org/wiki/Chymotrypsin chymotrypsin]. By modifying each of the catalytic residues separately and then testing the variant enzyme’s relative activity, it has been shown that mutation of any one of these residues dramatically reduces activity. The S124 alcohol’s nucleophilicity is inductively strengthened via deprotonation by H260 and E224, which allows the S124 residue to catalyze the reaction (Figure 4).
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[[Image:Ag85CCatalyticMechanism.jpg|800 px|center|thumb|'''Figure 4:''' Catalytic mechanism]]
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===Cys 209===
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<scene name='69/694218/Cysteine_209/2'>Cysteine 209</scene> is located near the Ag85C active site, but is not directly involved in the catalytic mechanism. However, formation of a functional catalytic triad relies on upon a <scene name='69/694220/C209/1'>Van der Waals interaction between C209 and the peptide bond between L232 and T231</scene>. The C209 facilitated interaction stabilizes a <scene name='69/694220/Alpha_9_helix/2'>kinked conformation of the α9 helix</scene> that promotes optimal interaction distances between catalytic residues.
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==Structures of Variant Enzymes==
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===Covalent Modification of Cys209===
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Ag85C has been crystallized following covalent inhibition by several thiol-modifying agents: ebselen, ''p''-chloromercuribenzoic acid, and iodoacetamide. Each of these thiol-reactive inhibitors covalently bound to C209 and caused a relaxation of the α9 helix. The resulting relaxed conformation of the helix significantly reduces or completely eliminates the enzymatic function of Ag85C.<ref name="Favrot2014"/>
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====Modification by Ebselen====
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[[Image:Ebselen_inhibition.jpeg|200 px|right|thumb|'''Figure 5:''' Pink: native enzyme; tan: ebselen-modified Ag85C; green: ebselen]]
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Ag85C can be inhibited by [http://en.wikipedia.org/wiki/Ebselen ebselen], which covalently bounds to the sulfur in C209. Ebselen is a thiol-modifying agent that serves as an electrophile for a C209 nucleophilic attack that results in sulfur-selenium bond formation. Crystallization of ebselen-modified Ag85C provides an explanation for the mechanism of inhibition. The addition of ebselen increases the distance between C209 and L232-T231, which effectively disrupts the interaction that holds the α9 helix in the active conformation. The disruption of this interaction causes the α9 helix to <scene name='69/694220/Inhibited_relaxed_helix/1'>relax</scene>. Furthermore, the bulk of ebselen creates steric hindrance with the α9 helix residues, which can be seen in '''Figure 5'''. Relaxation of the α9 helix due to ebselen modification moves E228 and H260, which now interacts with S148, instead of S124, in the active site. The displacement of these residues destroys the catalytic triad's charge relay, and as a result, the nucleophilicity of the S124 alcohol is not longer strengthened, which decreases catalytic activity.
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====Modification by ''p''-Chloromercuribenzoic acid====
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The thiol of C209 can also be covalently modified by [http://en.wikipedia.org/wiki/4-Chloromercuribenzoic_acid p-chloromercuribenzoic acid]. Similar to what is observed in Ag85C-ebselen, the alteration in <scene name='69/694218/4qdo/1'>Ag85C-Hg</scene> relaxes the kinked helix α-9 found in the native structure of the enzyme. The native structure is shown in green and the relaxed, modified structure is shown in blue. This conformational change again disrupts the hydrogen bonding network of the catalytic triad, resulting in a decrease to only 60% of the normal enzymatic function<ref name="Favrot2014"/>.
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===Mutation of Cys209===
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====E228Q====
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<scene name='69/694218/Ag85c-e228q/1'>Mutation of the glutamate component of the catalytic triad, E228, to glutamine</scene> decreased enzymatic activity to only 17% of the wild type<ref name="Favrot2014"/>. The α-9 helix is again relaxed in this variant. The mutated residue was shifted 4 angstroms from its original position in the native structure. Associated with this shift of Glu228 is the loss of hydrogen bonds between Ser124 and His260. The His260 residue takes on two conformations, <scene name='69/694218/4qdz/2'>in which it can hydrogen bond to either Ser124 or Ser148</scene>.
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{{clear}}
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====H260Q====
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<scene name='69/694218/Mutationh260q/1'>Mutation of the histidine component of the catalytic triad, H260, to glutamine</scene> also results in a shift in helix α-9 and <scene name='69/694218/Ag85c-hg/1'>prevents the hydrogen bond formation</scene> between residues His260 and Glu228, thus decreasing enzymatic activity.
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==Student Contributors==
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Benjamin Lancaster, Benjamin Zercher, Sarah Zimmerman, & Morgan Blake
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</StructureSection>
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== References ==
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<references/>
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Current revision

1CPX - Carboxypeptidase A from Bovine Pancreas

Introduction

is a metalloexopeptidase that hydrolytically cleaves peptides at the C-terminal peptide bond containing hydrophobic side chains. [1] Specifically, CPA is a Zn2+ metalloenzyme 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] From bovine pancreas, 1CPX has been crystallized in a new environment alongside two Zn2+ ions, one catalytic, the other inhibitory. The inhibitory Zn2+ ion in this more recent crystal structure not only prevents 1CPX from undergoing its hydrolysis mechanism; but also, this crystallographic data reveals a different conformation of the Tyr248 (GREEN LINK) residue suggesting alternative mechanistic behavior. [1] 1CPX consists of a single polypeptide chain that contains 307 amino acids. CPA proteins must first be activated by either trypsin or chymotrypsin in order to achieve an active form that serves its biological function.[1] This crystallographic data presents 1CPX activated by trypsin in its β-form and orthorhombic crystal form.


This is about 1CPX. This is a carboxypeptidase from bovine pancreas with two Zinc binding sites.This is about 1CPX. This is a carboxypeptidase from bovine pancreas with two Zinc binding sites.This is about 1CPX. This is a carboxypeptidase from bovine pancreas with two Zinc binding sites.This is about 1CPX. This is a carboxypeptidase from bovine pancreas with two Zinc binding sites.This is about 1CPX.

This is a carboxypeptidase from bovine pancreas with two Zinc binding sites.

This is a caption about 1CPX

Drag the structure with the mouse to rotate
Figure Legend
Figure Legend
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