Sandbox Reserved 1096: Difference between revisions

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The structure of the '''''apoenzyme''''' '''''apoPAD2''''' shows a stable head to tail dimer. The monomer is formed by 2 immunoglobulin-like domains and a C-term catalytic domain calcium binding site. There are six different calcium binding sites '''''(C1 to C6)''''', C2-5 are unoccupied in apoPAD2 but there is an electron density on C1 and C6 so those are occupied by calcium in the holoenzyme.

The structure of the '''''PAD2 Ca2+ complex''''' in 10mM of CaCl2 ( [[4n2b]]) differs from apoPAD2. Folding and 3D structure remain unchanged except C3-5 are occupied by Calcium cations when Ca2+ increases<ref name="ART1" />. The ''''apoPAD2'''' enzyme resolved structure shows 2 high-affinity Calcium binding sites that remain consistent. At 10mM Ca3-5 sites are occupied by Calcium cations and, even though there is still only one unoccupied site, the structure is not catalytically competent. This may be explained by the nucleophile active site C647 that is still 12 angstrom away from the catalytic center at 10mM. Yet, the 3 other key catalytic residues: D351, H471 and D473 are properly positioned to promote catalysis and, since they have the same conformation in both the apoenzyme and the holoenzyme, the complex structure PAD2+/Ca2+ represents an intermediate form between the 2 structures. This intermediate structure is stabilized by hydrogen bonds between R347 and Q350 in the active site. These bonds may also inhibit the movement of C647 into the substrate binding pocket<ref name="ART1" /> .

The structure of the '''''PAD2 Ca2+ complex''''' in 10mM of CaCl2 ([[4n2b]]) differs from apoPAD2. Folding and 3D structure remain unchanged except C3-5 are occupied by Calcium cations when Ca2+ increases<ref name="ART1" />. The ''''apoPAD2'''' enzyme resolved structure shows 2 high-affinity Calcium binding sites that remain consistent. At 10mM Ca3-5 sites are occupied by Calcium cations and, even though there is still only one unoccupied site, the structure is not catalytically competent. This may be explained by the nucleophile active site C647 that is still 12 angstrom away from the catalytic center at 10mM. Yet, the 3 other key catalytic residues: D351, H471 and D473 are properly positioned to promote catalysis and, since they have the same conformation in both the apoenzyme and the holoenzyme, the complex structure PAD2+/Ca2+ represents an intermediate form between the 2 structures. This intermediate structure is stabilized by hydrogen bonds between R347 and Q350 in the active site. These bonds may also inhibit the movement of C647 into the substrate binding pocket<ref name="ART1" /> .

To obtain the structure of '''''PAD2 holoenzyme'''''([[4n2c]]), a double mutant F221/F222A was engineered to prevent undesired interactions with neighboring hydrophobic pockets that could prevent binding of Calcium at Ca2 site. Structure of PAD2 F221/222A Ca2+ mutant was solved and studied showing an important electron density at all 6 calcium binding sites at 10mM Ca2+. All sites are now binding with calcium. Moreover, The active site cysteine, C647 points toward the catalytic center making the holoenzyme competent for catalysis. Calcium binding to Ca2 site was also found to cause R347 to move out of the active site while W348 moves in to form one wall of the substrate-binding pocket.

To obtain the structure of '''''PAD2 holoenzyme''''' ([[4n2c]]), a double mutant F221/F222A was engineered to prevent undesired interactions with neighboring hydrophobic pockets that could prevent binding of Calcium at Ca2 site. Structure of PAD2 F221/222A Ca2+ mutant was solved and studied showing an important electron density at all 6 calcium binding sites at 10mM Ca2+. All sites are now binding with calcium. Moreover, The active site cysteine, C647 points toward the catalytic center making the holoenzyme competent for catalysis. Calcium binding to Ca2 site was also found to cause R347 to move out of the active site while W348 moves in to form one wall of the substrate-binding pocket.

[[Image:PAD2.jpg|500px|right|thumb| Main transformations of apo and holo-enzymes that explain PAD2's activity. Obtained from https://doi.org/10.1021/cb500933j for educational use]]

Sandbox Reserved 1096: Difference between revisions

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 The structure of the '''''apoenzyme''''' '''''apoPAD2''''' shows a stable head to tail dimer. The monomer is formed by 2 immunoglobulin-like domains and a C-term catalytic domain calcium binding site. There are six different calcium binding sites '''''(C1 to C6)''''', C2-5 are unoccupied in apoPAD2 but there is an electron density on C1 and C6 so those are occupied by calcium in the holoenzyme.
-The structure of the '''''PAD2 Ca2+ complex''''' in 10mM of CaCl2 ( [[4n2b]]) differs from apoPAD2. Folding and 3D structure remain unchanged except C3-5 are occupied by Calcium cations when Ca2+ increases<ref name="ART1" />. The ''''apoPAD2'''' enzyme resolved structure shows 2 high-affinity Calcium binding sites that remain consistent. At 10mM Ca3-5 sites are occupied by Calcium cations and, even though there is still only one unoccupied site, the structure is not catalytically competent. This may be explained by the nucleophile active site C647 that is still 12 angstrom away from the catalytic center at 10mM. Yet, the 3 other key catalytic residues: D351, H471 and D473 are properly positioned to promote catalysis and, since they have the same conformation in both the apoenzyme and the holoenzyme, the complex structure PAD2+/Ca2+ represents an intermediate form between the 2 structures. This intermediate structure is stabilized by hydrogen bonds between R347 and Q350 in the active site. These bonds may also inhibit the movement of C647 into the substrate binding pocket<ref name="ART1" /> .
+The structure of the '''''PAD2 Ca2+ complex''''' in 10mM of CaCl2 ([[4n2b]]) differs from apoPAD2. Folding and 3D structure remain unchanged except C3-5 are occupied by Calcium cations when Ca2+ increases<ref name="ART1" />. The ''''apoPAD2'''' enzyme resolved structure shows 2 high-affinity Calcium binding sites that remain consistent. At 10mM Ca3-5 sites are occupied by Calcium cations and, even though there is still only one unoccupied site, the structure is not catalytically competent. This may be explained by the nucleophile active site C647 that is still 12 angstrom away from the catalytic center at 10mM. Yet, the 3 other key catalytic residues: D351, H471 and D473 are properly positioned to promote catalysis and, since they have the same conformation in both the apoenzyme and the holoenzyme, the complex structure PAD2+/Ca2+ represents an intermediate form between the 2 structures. This intermediate structure is stabilized by hydrogen bonds between R347 and Q350 in the active site. These bonds may also inhibit the movement of C647 into the substrate binding pocket<ref name="ART1" /> .
-To obtain the structure of '''''PAD2 holoenzyme'''''([[4n2c]]), a double mutant F221/F222A was engineered to prevent undesired interactions with neighboring hydrophobic pockets that could prevent binding of Calcium at Ca2 site. Structure of PAD2 F221/222A Ca2+ mutant was solved and studied showing an important electron density at all 6 calcium binding sites at 10mM Ca2+. All sites are now binding with calcium. Moreover, The active site cysteine, C647 points toward the catalytic center making the holoenzyme competent for catalysis. Calcium binding to Ca2 site was also found to cause R347 to move out of the active site while W348 moves in to form one wall of the substrate-binding pocket.
+To obtain the structure of '''''PAD2 holoenzyme''''' ([[4n2c]]), a double mutant F221/F222A was engineered to prevent undesired interactions with neighboring hydrophobic pockets that could prevent binding of Calcium at Ca2 site. Structure of PAD2 F221/222A Ca2+ mutant was solved and studied showing an important electron density at all 6 calcium binding sites at 10mM Ca2+. All sites are now binding with calcium. Moreover, The active site cysteine, C647 points toward the catalytic center making the holoenzyme competent for catalysis. Calcium binding to Ca2 site was also found to cause R347 to move out of the active site while W348 moves in to form one wall of the substrate-binding pocket.
 [[Image:PAD2.jpg|500px|right|thumb| Main transformations of apo and holo-enzymes that explain PAD2's activity. Obtained from https://doi.org/10.1021/cb500933j for educational use]]