Sandbox Reserved 1058: Difference between revisions
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==General Structure== | ==General Structure== | ||
DDAH-1’s <scene name='69/694225/Secondary_structure_colored/3'>secondary structure</scene> has a <scene name='69/694225/Prop_domains/2'>propeller-like fold</scene> which is characteristic of the superfamily of <span class="plainlinks">[https://en.wikipedia.org/wiki/Arginine:glycine_amidinotransferase L-arginine/glycine amidinotransferases]</span> <ref name="humm">Humm A, Fritsche E, Mann K, Göhl M, Huber R. Recombinant expression and isolation of human L-arginine:glycine amidinotransferase and identification of its active-site cysteine residue. Biochemical Journal. 1997 March 15;322(3):771-776. PMID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1218254/ 9148748]</span> doi:<span class="plainlinks">[http://www.biochemj.org/content/322/3/771 10.1042/bj3220771]</span></ref>. This five-stranded <span class="plainlinks">[https://en.wikipedia.org/wiki/Beta-propeller propeller]</span> contains five repeats of a ββαβ motif <ref name="frey" />. These motifs in DDAH form a <scene name='75/752351/Ddah_water_pore/12'>channel</scene> filled with water molecules (red spheres). Lys174 and Glu77 form a <scene name='75/752351/Ddah_salt_bridge/5'>salt bridge</scene> in the channel that forms the bottom of the <scene name='75/752351/Ddah_active_site/3'>active site</scene> shown here filled with water molecules. One side of the channel is a <scene name='75/752351/Ddah_water_pore/13'>water-filled pore</scene>, whereas the other side is the active site cleft <ref name="frey" />. | |||
===Lid Region=== | ===Lid Region=== | ||
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=====Important residues in Zinc Binding===== | =====Important residues in Zinc Binding===== | ||
It was found that Cys273, His172, Glu77, Asp78, and Asp 268 all <scene name='69/694225/Active_site6hbonds/3'>play a role</scene> in the binding of Zn(II). <scene name='69/694225/Cys273_zn/2'>Cys273</scene> directly coordinates with the Zn(II) ion in the active site while the other significant residues stabilize the ion via hydrogen bonding interactions with water molecules in the active site. Depending on pH, His172 can <scene name='69/694225/Active_site_9/2'> | It was found that Cys273, His172, Glu77, Asp78, and Asp 268 all <scene name='69/694225/Active_site6hbonds/3'>play a role</scene> in the binding of Zn(II). <scene name='69/694225/Cys273_zn/2'>Cys273</scene> directly coordinates with the Zn(II) ion in the active site while the other significant residues stabilize the ion via hydrogen bonding interactions with water molecules in the active site. Depending on pH, His172 can change conformation. At pH 9.0, DDAH-1 has been crystalized with <scene name='69/694225/Active_site_9/2'>His172</scene> in both conformations. Both of these conformations use the <span class="plainlinks">[https://en.wikipedia.org/wiki/Imidazole imidazole]</span> group to directly coordinate the Zn(II) ion. Cys273, which is conserved between bovine and humans, is the key active site residue that coordinates Zn(II) <ref name="frey" />. Zinc-cysteine complexes have been found to be important mediators of protein <span class="plainlinks">[https://en.wikipedia.org/wiki/Catalysis catalysis]</span>, regulation, and structure <ref name="pace">Pace NJ, Weerpana E. Zinc-binding cysteines: diverse functions and structural motifs. Biomolecules. 2014 June;4(2):419-434. PMCID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4101490/ 4101490]</span> doi:<span class="plainlinks">[http://www.mdpi.com/2218-273X/4/2/419/htm 10.3390/biom4020419]</span> </ref>. Cys273 and the water molecules stabilize the Zn(II) ion in a tetrahedral environment. The Zn(II) dissociation constant is 4.2 nM which is consistent with the nanomolar concentrations of Zn(II) in the cells, which provides more evidence for the regulatory use of Zn(II) by DDAH <ref name="pace" />. | ||
====Inhibitors==== | ====Inhibitors==== | ||
<scene name='75/752351/Ddah_l-homocysteine/3'>L-homocysteine</scene> and <scene name='75/752351/Ddah_with_l-citrulline/5'>L-citrulline</scene> bind in the active site in the same orientation as MMA and ADMA to create the same <span class="plainlinks">[https://en.wikipedia.org/wiki/Intermolecular_force intermolecular bonds]</span> between them and DDAH <ref name="frey" /> (Figure 6). L-citrulline is also a product of DDAH hydrolyzing ADMA and MMA, suggesting DDAH activity creates a <span class="plainlinks">[https://en.wikipedia.org/wiki/Negative_feedback negative feedback]</span> loop on itself (Figure 3). Both molecules enter the active site and cause DDAH to be in its closed lid formation. The α carbon on either molecule creates three salt bridges with DDAH: two with the guanidine group of Arg144 and one with the guanidine group Arg97. Another salt bridge is formed between the ligand and Asp72. The molecules are stabilized in the active site by <scene name='75/752351/Hbond_leu29/4'>four hydrogen bonds</scene>: α carbon-amino group of the ligand to main chain carbonyls of Val267 and Leu29. Hydrogen bonds also form between the side chains of Asp78 and Glu77 with the ureido group of L-citrulline. | <scene name='75/752351/Ddah_l-homocysteine/3'>L-homocysteine</scene> and <scene name='75/752351/Ddah_with_l-citrulline/5'>L-citrulline</scene> bind in the active site in the same orientation as MMA and ADMA to create the same <span class="plainlinks">[https://en.wikipedia.org/wiki/Intermolecular_force intermolecular bonds]</span> between them and DDAH <ref name="frey" /> (Figure 6). L-citrulline is also a product of DDAH hydrolyzing ADMA and MMA, suggesting DDAH activity creates a <span class="plainlinks">[https://en.wikipedia.org/wiki/Negative_feedback negative feedback]</span> loop on itself (Figure 3). Both molecules enter the active site and cause DDAH to be in its closed lid formation. The α carbon on either molecule creates three <scene name='75/752351/Hbond_leu29/7'>salt bridges</scene> salt bridges with DDAH: two with the guanidine group of Arg144 and one with the guanidine group on Arg97. Another salt bridge is formed between the ligand and Asp72. The molecules are stabilized in the active site by <scene name='75/752351/Hbond_leu29/4'>four hydrogen bonds</scene>: α carbon-amino group of the ligand to main chain carbonyls of Val267 and Leu29. Hydrogen bonds also form between the side chains of Asp78 and Glu77 with the ureido group of L-citrulline. | ||
Like L-homocysteine and L-citrulline, <scene name='75/752351/Ddah_s-nitroso-l-homocysteine/4'>S-nitroso-L-homocysteine</scene> binds and the lid region of DDAH is closed (Figure 6). When DDAH reacts with S-nitroso-L-homocysteine, a covalent product, N-thiosulfximide exist in the active site because of its binding to Cys273. N-thiosulfximide is stabilized by several salt bridges and hydrogen bonds. Arg144 and Arg97 stabilize the α carbon-carbonyl group via salt bridges, and Leu29, Val267, and Asp72 stabilize the α carbon-amino group by forming hydrogen bonds <ref name="frey" />. | Like L-homocysteine and L-citrulline, <scene name='75/752351/Ddah_s-nitroso-l-homocysteine/4'>S-nitroso-L-homocysteine</scene> binds and the lid region of DDAH is closed (Figure 6). When DDAH reacts with S-nitroso-L-homocysteine, a covalent product, N-thiosulfximide exist in the active site because of its binding to Cys273. N-thiosulfximide is stabilized by several salt bridges and hydrogen bonds. Arg144 and Arg97 stabilize the α carbon-carbonyl group via salt bridges, and Leu29, Val267, and Asp72 stabilize the α carbon-amino group by forming hydrogen bonds <ref name="frey" />. | ||
[[Image:L-citrulline, L-homocysteine, and S-nitroso-L-homocysteine.jpg|500px|center|thumb|'''Figure 6.''' Structures of DDAH inhibitors.]] | [[Image:L-citrulline, L-homocysteine, and S-nitroso-L-homocysteine.jpg|500px|center|thumb|'''Figure 6.''' Structures of DDAH inhibitors.]] | ||