Introduction

Dimethylarginine Dimethyaminohydrolase (commonly known as DDAH) is a member of the hydrolase family of enzymes which use water to break down molecules (palm). Specifically, DDAH is a nitric oxide synthase (NOS) regulator. Its metabolizes free arginine derivatives, namely NѠ,NѠ-dimethyl-L-arginine (ADMA) and NѠ-methyl-L-arginine (MMA) which competitively inhibit NOS (tran). DDAH converts MMA and ADMA to L-citrulline and mono- or dimethylamine (frey). DDAH is expressed in the cytosol of cells in humans, mice, rates, sheep, cattle, and bacteria (palm). DDAH activity has been localized mainly to the brain, kidney, pancreas, and liver in these organisms. If DDAH is overexpressed, NOS can be activated (Frey). ADMA and MMA can inhibit the synthesis of NO by competitively inhibiting all three kinds of NOS (endothelial, neuronal, and inducible) (frey). 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 (Janssen). NO is made by NOS creating L-citrulline from L-arginine (Frey). In humans, many diseases can come from improper control of NO levels including diabetes mellitus and hypertension. Current research has identified several inhibitors of DDAH which could be important in fighting diseases involving irregular NO levels (frey).

General Structure

Lid Region

Amino acids 25-36 of DDAH constitute the loop region of the protein (frey). This is more commonly known as the lid region. The lid is what allows the active site to be exposed to substate binding or not. Studies have shown crystal structures of the lid at open and closed conformations. In the open conformation, the lid forms an alpha helix and the amino acid Leu29 is moved so it does not interact with the active site. This allows the active site to be vulnerable to attack. This lid region is very flexible. This open conformation has been shown when DDAH had been crystalized when Zn2+ was bound at pH 6.3. There is a closed form which has been observed with Zn2+ binding at pH 9.0 and in the unliganded enzyme. When the lid is closed, a specific hydrogen bond can form between the Leu29 carbonyl and the amino group on bound molecule. This stabilizes this complex. The Leu29 is then blocking the active site entrance (frey). Opening and closing the lid takes place faster than the actual reaction in the active site (Rasheed). 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 (Rasheed). The specific residues in the lid region are different in different organisms (Frey). 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 (Rasheed). Different isoforms from the same species can have differences in lid regions as well (frey). DDAH-2 has a negatively charged lid while DDAH-1 has a positively charged lid (frey).

Active Site

The normal DDAH regulation mechanism depends on the presence of Cys249 in the active site that acts as a nucleophile in the mechanism (Stone). 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 His162 protonates the leaving group in this reaction and generates hydroxide to hydrolyze the intermediate formed in the reaction. 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 Histidine162 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 (stone).

Zn2+ Bound to the Active Site

Figure Legend

Medical Relevancy

Excess Nitric Oxide Effects

Inhibitors

Different Isoforms

DDAH has two main isoforms (frey). DDAH-1 colocalizes with nNOS (neuronal NOS). This enzyme is found mainly in the brain and kidney of organisms (tran). DDAH-2 is found in tissues with eNOS (endothelial NOS) (frey). DDAH-2 localization has been found in the heart, kidney, and placenta (tran). Additionally, studies show that DDAH-2 is expressed in iNOS containing immune tissues (inducible NOS) (frey). 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. 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 (frey).

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