Carboxypeptidase A: Difference between revisions
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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: | 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. | # Three S1 subsite residues (<scene name='69/694222/3cpas1subsiteresidues1/2'>Asn144, Arg145, and Tyr248</scene>) and the <scene name='69/694222/3cpahydrophobicpocketresidues/3'>hydrophobic S1' subsite</scene> recognize the C-terminus of the polypeptide substrate. | ||
# After aiding in the recognition of the substrate, Tyr248 "caps" the binding pocket. | # After aiding in the recognition of the substrate, <scene name='69/694222/3cpadeeppocket2/1'>Tyr248 "caps" the binding pocket</scene>. | ||
# The catalytic Zn<sup>2+</sup> 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. | # The catalytic Zn<sup>2+</sup> 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 [http://en.wikipedia.org/wiki/Deprotonation deprotonated] by Glu270 ([http://bio.libretexts.org/Core/Biochemistry/Catalysis/METHODS_OF_CATALYSIS/General_Acid%2F%2FBase_Catalysis 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 [http://en.wikipedia.org/wiki/Nucleophile nucleophile] and attacks this carbon to generate a [http://goldbook.iupac.org/T06289.html tetrahedral intermediate] stabilized by both the Zn<sup>2+</sup> ion and surrounding positive charges of S1 subsite residues. | # A water molecule, which has been [http://en.wikipedia.org/wiki/Deprotonation deprotonated] by Glu270 ([http://bio.libretexts.org/Core/Biochemistry/Catalysis/METHODS_OF_CATALYSIS/General_Acid%2F%2FBase_Catalysis 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 [http://en.wikipedia.org/wiki/Nucleophile nucleophile] and attacks this carbon to generate a [http://goldbook.iupac.org/T06289.html tetrahedral intermediate] stabilized by both the Zn<sup>2+</sup> ion and surrounding positive charges of S1 subsite residues. | ||
# The peptide bond is cleaved through an addition-elimination step. | # The peptide bond is cleaved through an addition-elimination step. | ||
# The Glu270 base catalyst is regenerated through a final [http://www.masterorganicchemistry.com/tips/proton-transfer/ proton transfer] with the nitrogen atom of the former C-terminal peptide bond. | # The <scene name='69/694222/3cpas1subsiteglu270/2'>Glu270 base catalyst</scene> is regenerated through a final [http://www.masterorganicchemistry.com/tips/proton-transfer/ 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. | # 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. | ||
[[Image:Proteopedia Reaction Mechanism Graphic.jpg|750 px|thumb|left|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 Zn<sup>2+</sup> 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).<ref name="CPA2" /> ]] | [[Image:Proteopedia Reaction Mechanism Graphic.jpg|750 px|thumb|left|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 Zn<sup>2+</sup> 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).<ref name="CPA2" /> ]] | ||