Sandbox Reserved 1058: Difference between revisions

DDAH’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/9'>channel</scene> filled with water molecules (red spheres). Lys174 and Glu77 form a <scene name='75/752351/Ddah_salt_bridge/4'>salt bridge</scene> in the channel that forms the bottom of the <scene name='75/752351/Ddah_active_site/1'>active site</scene> for the protein. One side of the channel is a <scene name='75/752351/Ddah_water_pore/3'>water-filled pore</scene>, whereas the other side is the active site cleft <ref name="frey" />.

<scene name='75/752351/Lid_focus/1'>loop region</scene> of the protein which is more commonly known as the lid region <ref name="frey" />. The lid is what allows the active site to be exposed to substrate binding or not. Studies have shown crystal structures of the lid at <scene name='69/694225/Open_surface/5'>open</scene>and <scene name='69/694225/Closed_surface/4'>closed</scene> conformations. In the open conformation, the lid forms an alpha helix and the amino acid <scene name='69/694225/Lid_helix/1'>Leu29</scene> is moved so it does not interact with the active site. This allows the active site to be vulnerable to attack. When the lid is closed, a specific <scene name=’75/752351/Hbond_leu29/1’>hydrogen bond</scene> can form between the Leu29 carbonyl and the amino group on bound molecule. This stabilizes this complex. The Leu29 is then <scene name='69/694225/Closed_lid_zn9/3'>blocking</scene> the active site entrance <ref name="frey" />. Opening and closing the lid takes place faster than the actual reaction in the active site <ref name="rasheed">Rasheed M, Richter C, Chisty LT, Kirkpatrick J, Blackledge M, Webb MR, Driscoll PC. Ligand-dependent dynamics of the active site lid in bacterial Dimethyarginine Dimethylaminohydrolase. Biochemistry. 2014 Feb 18;53:1092-1104. PMCID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945819/ PMC3945819]</span> doi:<span class="plainlinks">[http://pubs.acs.org/doi/abs/10.1021/bi4015924 10.1021/bi4015924]</span></ref>. This suggests that the <span class="plainlinks">[https://en.wikipedia.org/wiki/Rate-determining_step rate-limiting step]</span> of this reaction is not the lid movement but is the actual chemistry happening to the substrate in the active site of DDAH <ref name="rasheed" />.

The normal DDAH regulation <span class="plainlinks">[https://en.wikipedia.org/wiki/Reaction_mechanism mechanism]</span> depends on the presence of <scene name='75/752351/Ddah_active_site/2'>Cys 249</scene> in the active site that acts as a <span class="plainlinks">[https://en.wikipedia.org/wiki/Nucleophile nucleophile]</span> in the mechanism <ref name="stone">Stone EM, Costello AL, Tierney DL, Fast W. Substrate-assisted cysteine deprotonation in the mechanism of Dimethylargininase (DDAH) from Pseudomonas aeruginosa. Biochemistry. 2006 May 2;45(17):5618-5630. PMID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pubmed/16634643 16634643]</span> doi:<span class="plainlinks">[http://pubs.acs.org/doi/abs/10.1021/bi052595m 10.1021/bi052595m]</span></ref> (Figure 3). The Cys249 is used to attack the <span class="plainlinks">[https://en.wikipedia.org/wiki/Guanidine guanidinium]</span> carbon on the substrate that is held in the active site via hydrogen bonds. This is followed by collapsing the tetrahedral product to get rid of the <span class="plainlinks">[https://en.wikipedia.org/wiki/Alkylamines alkylamine]</span> leaving group. A <span class="plainlinks">[https://en.wikipedia.org/wiki/Isothiouronium thiouronium]</span> intermediate is then formed with <span class="plainlinks">[https://en.wikipedia.org/wiki/Orbital_hybridisation sp² hybridization]</span>. This intermediate is hydrolyzed to form citrulline. The <scene name='75/752351/Ddah_active_site_his162/1'>His162</scene> protonates the leaving group in this reaction and generates hydroxide to hydrolyze the intermediate formed in the reaction (Figure 3). L-citrulline leaves the active site when the lid opens. The amines can either leave through the entrance to the active site or through a pore made by movement of Glu77 and Lys174 <ref name="frey" />. Studies suggest that Cys249 is neutral until binding of guanidinium near Cys249 decreases Cys249’s <span class="plainlinks">[https://en.wikipedia.org/wiki/Acid_dissociation_constant pKa]</span> and deprotonates the thiolate to activate the nucleophile. Other studies suggest that the Cys249 and an active site His162 form an <span class="plainlinks">[https://en.wikipedia.org/wiki/Intimate_ion_pair ion pair]</span> to deprotonate the thiolate. Cys249 and His162 can also form a binding site for inhibitors to bind to which stabilizes the thiolate. This is important in regulating NO activity in organisms and designing drugs to inhibit this enzyme <ref name="stone" />.

There is a channel in the center of the protein that is closed by a <scene name='75/752351/Ddah_salt_bridge/3'>salt bridge</scene> connecting Glu77 and Lys174 <ref name="frey" />. This salt bridge constitutes the bottom of the active site. There is a pore containing water on one side of the channel. This pore is <scene name='75/752351/Ddah_water_pore/10'>delineated</scene> by the first β strand of each of the five propeller blades. The water in the water-filled pore forms hydrogen bonds to <scene name=’75/752351/Ddah_water_pore/7’>His172 and Ser175</scene>. The other side of the channel is the active site. Short loop regions and a helical structure define the outward boundaries of this site.

Active sites of DDAH from different organisms are similar. Amino acids involved in the chemical mechanism of creating products are also <scene name='69/694225/Evolutionary_conservation/2'>conserved</scene> (Figure 4).

In DDAH, <span class="plainlinks">[https://en.wikipedia.org/wiki/Zinc (Zn(II))]</span> acts as an endogenous inhibitor and prevents normal NOS activity <ref name="frey" /> (Figure 5). The Zn(II)-binding site is located inside the protein’s active site, which makes it a <span class="plainlinks">[https://en.wikipedia.org/wiki/Competitive_inhibition competitive inhibitor]</span>. When bound, Zn(II) blocks the entrance of any other substrate. An open conformation of the lid region has been shown when DDAH had been <span class="plainlinks">[https://en.wikipedia.org/wiki/Crystallization crystallized]</span> when Zn(II) was bound at <scene name='69/694225/Active_site6/1'>pH 6.3</scene> (Figure 5). There is a closed form which has been observed with Zn(II) binding at <scene name='69/694225/Active_site_9/1'>pH 9.0</scene> and in the unliganded enzyme (Figure 5).

It was found that Cys273, His172, Glu77, Asp78, and Asp 268 all <scene name='69/694225/Active_site6hbonds/2'>play a role</scene> in the binding of Zn(II). <scene name='69/694225/Cys273_zn/1'>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_9/1'>change conformation</scene> and 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" />.

<scene name='75/752351/Ddah_l-homocysteine/2'>L-homocysteine</scene> and <scene name='75/752351/Ddah_with_L-citrulline/4'>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 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. 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_leu29/2'>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/3'>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" />.