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Dimethylarginine DimethylaminohydrolaseDimethylarginine Dimethylaminohydrolase

Introduction

Dimethylarginine Dimethyaminohydrolase EC 3.5.3.18 (commonly known as DDAH) is a member of the hydrolase family of enzymes which use water to break down molecules ^[1]. Additionally, DDAH is a nitric oxide synthase (NOS) regulator. It metabolizes free arginine derivatives, namely N^Ѡ,N^Ѡ-dimethyl-L-arginine (ADMA) and N^Ѡ-methyl-L-arginine (MMA) which competitively inhibit NOS ^[2]. DDAH converts MMA or ADMA to two products: L-citrulline and an amine ^[3] (Figure 1). DDAH is expressed in the cytosol of cells in humans, mice, rates, sheep, cattle, and bacteria ^[1]. DDAH activity has been localized mainly to the brain, kidney, pancreas, and liver in these organisms. Presented in this page is information from DDAH isoform 1 (DDAH-1); however, there are two different isoforms ^[3].

Different Isoforms

DDAH has two main isoforms ^[3]. DDAH-1 colocalizes with nNOS (neuronal NOS). This enzyme is found mainly in the brain and kidney of organisms ^[2]. DDAH-2 is found in tissues with eNOS (endothelial NOS) ^[3]. DDAH-2 localization has been found in the heart, kidney, and placenta ^[2]. Additionally, studies show that DDAH-2 is expressed in iNOS containing immune tissues (inducible NOS) ^[3]. Both of the isoforms have conserved residues that are involved in the catalytic mechanism of DDAH (Cys, Asp, and His). The differences between the isoforms is in the substrate binding residues and the lid region residues. DDAH-1 has a positively charged lid region while DDAH-2 has negatively charged lid. In total, three salt bridge differ between DDAH-1 and DDAH-2 isoforms.

General Structure

DDAH’s has a which is characteristic of the superfamily of L-arginine/glycine amidinotransferases ^[4]. This five-stranded propeller contains five repeats of a ββαβ motif ^[3]. These motifs in DDAH form a filled with water molecules (red spheres). Lys174 and Glu77 form a in the channel that forms the bottom of the for the protein. One side of the channel is a , whereas the other side is the active site cleft ^[3].

Lid Region

Amino acids 25-36 of DDAH constitute the flexible

of the protein which is more commonly known as the lid region ^[3]. 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 and conformations. In the open conformation, the lid forms an alpha helix and the amino acid 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 can form between the Leu29 carbonyl and the amino group on bound molecule. This stabilizes this complex. The Leu29 is then the active site entrance ^[3]. Opening and closing the lid takes place faster than the actual reaction in the active site ^[5]. This suggests that the rate-limiting step of this reaction is not the lid movement but is the actual chemistry happening to the substrate in the active site of DDAH ^[5].

Lid Region Conservation

The specific residues in the lid region are different in different organisms ^[3] (Figure 2). The only consistent similarity is a conserved leucine residue in this lid that function to hydrogen bond with the ligand bound to the active site in DDAH-1 but not in DDAH-2 ^[5] (Figure 2). Different isoforms from the same species can have differences in lid regions as well ^[3]. DDAH-2 has a negatively charged lid while DDAH-1 has a positively charged lid ^[3].

Active Site

The normal DDAH regulation mechanism depends on the presence of in the active site that acts as a nucleophile in the mechanism ^[6] (Figure 3). The Cys249 is used to attack the guanidinium 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 alkylamine leaving group. A thiouronium intermediate is then formed with sp² hybridization. This intermediate is hydrolyzed to form citrulline. The 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 ^[3]. Studies suggest that Cys249 is neutral until binding of guanidinium near Cys249 decreases Cys249’s pKa and deprotonates the thiolate to activate the nucleophile. Other studies suggest that the Cys249 and an active site His162 form an ion pair 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 ^[6].

Channel with Salt Bridge and Water Pore

There is a channel in the center of the protein that is closed by a connecting Glu77 and Lys174 ^[3]. 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 by the first β strand of each of the five propeller blades. The water in the water-filled pore forms hydrogen bonds to . 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 Site Conservation

Active sites of DDAH from different organisms are similar. Amino acids involved in the chemical mechanism of creating products are also (Figure 4).

**Figure 4.** Color key for DDAH conservation

Zn(II) Bound to the Active Site

In DDAH, (Zn(II)) acts as an endogenous inhibitor and prevents normal NOS activity ^[3] (Figure 5). The Zn(II)-binding site is located inside the protein’s active site, which makes it a competitive inhibitor. 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 crystallized when Zn(II) was bound at (Figure 5). There is a closed form which has been observed with Zn(II) binding at and in the unliganded enzyme (Figure 5).

Important residues in Zinc Binding

It was found that Cys273, His172, Glu77, Asp78, and Asp 268 all in the binding of Zn(II). 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 and use the imidazole 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) ^[3]. Zinc-cysteine complexes have been found to be important mediators of protein catalysis, regulation, and structure ^[7]. 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 ^[7].

Inhibitors

and bind in the active site in the same orientation as MMA and ADMA to create the same intermolecular bonds between them and DDAH ^[3] (Figure 6). L-citrulline is a product of DDAH hydrolyzing ADMA and MMA, suggesting DDAH activity creates a negative feedback 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 : 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, 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 ^[3].

**Figure 6.** Structures of DDAH inhibitors.

Clinical Relevance

DDAH works to hydrolyze MMA and ADMA ^[3]. Both MMA and ADMA competitively inhibit NO synthesis by inhibiting Nitric Oxide Synthase (NOS). NO is made by NOS creating L-citrulline from L-arginine ^[3]. If DDAH is overexpressed, NOS can be activated ^[3]. ADMA and MMA can inhibit the synthesis of NO by competitively inhibiting all three kinds of NOS (endothelial, neuronal, and inducible) ^[3]. Underexpression or inhibition of DDAH decreases NOS activity and NO levels will decrease. Because of nitric oxide’s (NO) role in signaling and defense, NO levels in an organism must be regulated to reduce damage to cells ^[8]. NO is an important signaling and effector molecule in neurotransmission, bacterial defense, and regulation of vascular tone ^[9]. Because NO is highly toxic, freely diffusible across membranes, and its radical form is fairly reactive, cells must maintain a large control on concentrations by regulating NOS activity and the activity of enzymes such as DDAH that have an indirect effect of the concentration of NO ^[10]. An imbalance of NO contributes to several diseases. Low NO levels, potentially caused by low DDAH activity and therefore high MMA and ADMA concentrations, have been implicated with diseases such as uremia, chronic heart failure, atherosclerosis, and hyperhomocysteinemia ^[11]. High levels of NO have been involved with diseases such as septic shock, migraine, inflammation, and neurodegenerative disorders ^[12]. Because of the effects on NO levels and known inhibitors to DDAH, regulation of DDAH may be an effective way to regulate NO levels therefore treating these diseases ^[3]. Additionally, researchers can take advantage of the fact that there are two different isoforms of this enzyme and create drugs that target one isoform over another to control NO levels in specific tissues in the body ^[3].

ReferencesReferences

↑ ^1.0 ^1.1 Palm F, Onozato ML, Luo Z, Wilcox CS. Dimethylarginine dimethylaminohydrolase (DDAH): expression, regulation, and function in the cardiovascular and renal systems. American Journal of Physiology. 2007 Dec 1;293(6):3227-3245. PMID:17933965 doi:10.1152/ajpheart.00998.2007
↑ ^2.0 ^2.1 ^2.2 Tran CTL, Leiper JM, Vallance P. The DDAH/ADMA/NOS pathway. Atherosclerosis Supplements. 2003 Dec;4(4):33-40. PMID:14664901 doi:10.1016/S1567-5688(03)00032-1
↑ ^3.00 ^3.01 ^3.02 ^3.03 ^3.04 ^3.05 ^3.06 ^3.07 ^3.08 ^3.09 ^3.10 ^3.11 ^3.12 ^3.13 ^3.14 ^3.15 ^3.16 ^3.17 ^3.18 ^3.19 ^3.20 ^3.21 ^3.22 ^3.23 Frey D, Braun O, Briand C, Vasak M, Grutter MG. Structure of the mammalian NOS regulator dimethylarginine dimethylaminohydrolase: a basis for the design of specific inhibitors. Structure. 2006 May;14(5):901-911. PMID:[1] doi:10.1016/j.str.2006.03.006
↑ 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:9148748 doi:10.1042/bj3220771
↑ ^5.0 ^5.1 ^5.2 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:PMC3945819 doi:10.1021/bi4015924
↑ ^6.0 ^6.1 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:16634643 doi:10.1021/bi052595m
↑ ^7.0 ^7.1 Pace NJ, Weerpana E. Zinc-binding cysteines: diverse functions and structural motifs. Biomolecules. 2014 June;4(2):419-434. PMCID:4101490 doi:10.3390/biom4020419
↑ Janssen W, Pullamsetti SS, Cooke J, Weissmann N, Guenther A, Schermuly RT. The role of dimethylarginine dimethylaminohydrolase (DDAH) in pulmonary fibrosis. The Journal of Pathology. 2012 Dec 12;229(2):242-249. Epub 2013 Jan. PMID:23097221 doi:10.1002/path.4127
↑ Colasanti M, Suzuki H. The dual personality of NO. ScienceDirect. 2000 Jul 1;21(7):249-252. PMID:10979862 doi:10.1016/S0165-6147(00)01499-1
↑ Rassaf T, Feelisch M, Kelm M. Circulating NO pool: assessment of nitrite and nitroso species in blood and tissues. Free Rad. Biol. Med. 2004 Feb 15;36(4):413-422. PMID:14975444 doi:10.1016/j.freeradbiomed.2003.11.011
↑ Tsao PS, Cooke JP. Endothelial alterations in hypercholesterolemia: more than simply vasodilator dysfunction. Journal of Cardiovascular Pharmacology. 1998;32(3):48-53. PMID:9883748
↑ Vallance P, Leiper J. Blocking NO synthesis: how, where and why? Nat. Rev. Drug Discov. 2002 Dec;1(12):939-950. PMID:12461516 doi:10.1038/nrd960

Student ContributorsStudent Contributors

Natalie Van Ochten
Kaitlyn Enderle
Colton Junod

3D Structures of Dimethylarginine Dimethylaminohydrolase3D Structures of Dimethylarginine Dimethylaminohydrolase

2C6Z L-citrulline bound to isoform 1

2CI1 S-nitroso-L-homocysteine bound to isoform 1

2CI3 crystal form 1

2CI4 crystal form 2

2CI5 L-homocysteine bound to isoform 1

2CI6 Zn (II) bound at low pH to isoform 1

2CI7 Zn (II) bound at high pH to isoform 1

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA, Perry Rabin, Elizabeth Hughes, Sydney Pate, Geoffrey C. Hoops, Colton Junod, Natalie Van Ochten

[palm-1] 1.0 ^1.1 Palm F, Onozato ML, Luo Z, Wilcox CS. Dimethylarginine dimethylaminohydrolase (DDAH): expression, regulation, and function in the cardiovascular and renal systems. American Journal of Physiology. 2007 Dec 1;293(6):3227-3245. PMID:17933965 doi:10.1152/ajpheart.00998.2007

[tran-2] 2.0 ^2.1 ^2.2 Tran CTL, Leiper JM, Vallance P. The DDAH/ADMA/NOS pathway. Atherosclerosis Supplements. 2003 Dec;4(4):33-40. PMID:14664901 doi:10.1016/S1567-5688(03)00032-1

[frey-3] 3.00 ^3.01 ^3.02 ^3.03 ^3.04 ^3.05 ^3.06 ^3.07 ^3.08 ^3.09 ^3.10 ^3.11 ^3.12 ^3.13 ^3.14 ^3.15 ^3.16 ^3.17 ^3.18 ^3.19 ^3.20 ^3.21 ^3.22 ^3.23 Frey D, Braun O, Briand C, Vasak M, Grutter MG. Structure of the mammalian NOS regulator dimethylarginine dimethylaminohydrolase: a basis for the design of specific inhibitors. Structure. 2006 May;14(5):901-911. PMID:[1] doi:10.1016/j.str.2006.03.006

[humm-4] 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:9148748 doi:10.1042/bj3220771

[rasheed-5] 5.0 ^5.1 ^5.2 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:PMC3945819 doi:10.1021/bi4015924

[stone-6] 6.0 ^6.1 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:16634643 doi:10.1021/bi052595m

[pace-7] 7.0 ^7.1 Pace NJ, Weerpana E. Zinc-binding cysteines: diverse functions and structural motifs. Biomolecules. 2014 June;4(2):419-434. PMCID:4101490 doi:10.3390/biom4020419

[janssen-8] Janssen W, Pullamsetti SS, Cooke J, Weissmann N, Guenther A, Schermuly RT. The role of dimethylarginine dimethylaminohydrolase (DDAH) in pulmonary fibrosis. The Journal of Pathology. 2012 Dec 12;229(2):242-249. Epub 2013 Jan. PMID:23097221 doi:10.1002/path.4127

[colasanti-9] Colasanti M, Suzuki H. The dual personality of NO. ScienceDirect. 2000 Jul 1;21(7):249-252. PMID:10979862 doi:10.1016/S0165-6147(00)01499-1

[rassaf-10] Rassaf T, Feelisch M, Kelm M. Circulating NO pool: assessment of nitrite and nitroso species in blood and tissues. Free Rad. Biol. Med. 2004 Feb 15;36(4):413-422. PMID:14975444 doi:10.1016/j.freeradbiomed.2003.11.011

[tsao-11] Tsao PS, Cooke JP. Endothelial alterations in hypercholesterolemia: more than simply vasodilator dysfunction. Journal of Cardiovascular Pharmacology. 1998;32(3):48-53. PMID:9883748

[vallance-12] Vallance P, Leiper J. Blocking NO synthesis: how, where and why? Nat. Rev. Drug Discov. 2002 Dec;1(12):939-950. PMID:12461516 doi:10.1038/nrd960

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@@ Line 10: / Line 10: @@
 ==General Structure==
-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" />.
+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/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> for the protein. 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===
 Amino acids 25-36 of DDAH constitute the flexible
-<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" />.
+<scene name='75/752351/Lid_focus/2'>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/8'>open</scene> and <scene name='69/694225/Closed_surface/5'>closed</scene> conformations. In the open conformation, the lid forms an alpha helix and the amino acid <scene name='69/694225/Lid_helix/2'>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_leu29/3'>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" />.
 ====Lid Region Conservation====
@@ Line 21: / Line 21: @@
 ===Active Site===
-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 sp<sup>2</sup> 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" />.
+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/4'>Cys249</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 sp<sup>2</sup> hybridization]</span>. This intermediate is hydrolyzed to form citrulline. The <scene name='75/752351/Ddah_active_site_his162/2'>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" />.
 [[Image:The Normal DDAH Mechanism.jpg|800px|center|thumb|'''Figure 3.''' The normal mechanism of DDAH highlighting important residues involved.]]
 ====Channel with Salt Bridge and Water Pore====
-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.
+There is a channel in the center of the protein that is closed by a <scene name='75/752351/Ddah_salt_bridge/6'>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/14'>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/15'>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 Site Conservation====
-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).
+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/3'>conserved</scene> (Figure 4).
 [[Image:ColorKey ConSurf NoYellow NoGray.gif|400px|right|thumb|'''Figure 4.''' Color key for DDAH conservation]]
 ====Zn(II) Bound to the Active Site====
-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).
+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_site6/2'>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_9/3'>pH 9.0</scene> and in the unliganded enzyme (Figure 5).
 [[Image:Zn(II) bound at differing pH values.jpg|500 px|center|thumb|'''Figure 5.''' Zn(II) bound to the active site of DDAH at differing pH values. A) Zn(II) bound at pH 9.0 showing the channel of DDAH. B) Zn(II) bound at 9.0 showing the closed conformation lid with Leu29 blocking the active site. C) Zn(II) bound at pH 6.3 showing the channel of DDAH. D) Zn(II) bound at pH 6.3 showing the open lid conformation with Leu29 away from the active site.]]
 =====Important residues in Zinc Binding=====
-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" />.
+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'>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" />.
 ====Inhibitors====
-<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.
+<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 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_leu29/4'>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/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" />.
+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.]]

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Revision as of 05:07, 21 April 2017

Dimethylarginine DimethylaminohydrolaseDimethylarginine Dimethylaminohydrolase

Contents

Introduction

Different Isoforms