User:Natalie Van Ochten/Sandbox 1
From Proteopedia
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The normal DDAH regulation mechanism depends on the presence of Cys249 in the active site that acts as a nucleophile in the mechanism <ref name="stone"[doi:10.1021/bi052595m]</ref>. 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 <ref name="stone" />. | The normal DDAH regulation mechanism depends on the presence of Cys249 in the active site that acts as a nucleophile in the mechanism <ref name="stone"[doi:10.1021/bi052595m]</ref>. 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 <ref name="stone" />. | ||
There is a channel in the center of the protein that is closed by a salt bridge connecting Glu77 and Lys174. 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 delineated by the first β strand of each of the five propeller blades. The water in the water-filled pore forms hydrogen bonds to His172 and Ser175. 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 is similar. Amino acids involved in the chemical mechanism of creating products are also conserved. Amino acids in the lid region are not conserved except for a Leucine amino acid. | There is a channel in the center of the protein that is closed by a salt bridge connecting Glu77 and Lys174. 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 delineated by the first β strand of each of the five propeller blades. The water in the water-filled pore forms hydrogen bonds to His172 and Ser175. 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 is similar. Amino acids involved in the chemical mechanism of creating products are also conserved. Amino acids in the lid region are not conserved except for a Leucine amino acid. | ||
| - | When MMA or ADMA bind in the active site, they are broken down into L-citrulline and amines (Figure 1). 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 | + | When MMA or ADMA bind in the active site, they are broken down into L-citrulline and amines (Figure 1). 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. |
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==Medical Relevancy== | ==Medical Relevancy== | ||
| - | DDAH works to hydrolyze MMA and ADMA. Both MMA and ADMA competitively inhibit NO synthesis by inhibiting Nitric Oxide Synthase (NOS). 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. 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. High levels of NO have been involved with diseases such as septic shock, migraine, inflammation, and neurodegenerative disorders. 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 the diseases. | + | DDAH works to hydrolyze MMA and ADMA. Both MMA and ADMA competitively inhibit NO synthesis by inhibiting Nitric Oxide Synthase (NOS). NO is an important signaling and effector molecule in neurotransmission, bacterial defense, and regulation of vascular tone (1). 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 (2). 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 (3). High levels of NO have been involved with diseases such as septic shock, migraine, inflammation, and neurodegenerative disorders (4). 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 the diseases. |
==Inhibitors== | ==Inhibitors== | ||
Revision as of 12:25, 28 March 2017
Dimethylarginine Dimethylaminohydrolase
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References
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 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:16698551 doi:10.1016/j.str.2006.03.006
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/full
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
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 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
