The catalytic mechanism of dimethylarginine dimethylaminohydrolase (DDAH) from pseudomonas aeruginosa

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2006

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Stone, Everett Monroe

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Abstract

Dimethylarginine dimethylaminohydrolase (DDAH) catalyzes the hydrolysis of Nw–methyl–L–arginine (NMMA) and Nw,Nw –methyl–L–arginine (ADMA) to L-citrulline and methylamine or dimethylamine, respectively. ADMA and NMMA are endogenous inhibitors of nitric oxide synthase (NOS) in mammals. DDAH therefore partially regulates NOS activity, making it an attractive therapeutic target in disease states involving overproduction of nitric oxide. Understanding the mechanism of DDAH is important to inhibitor design and elucidating its physiological function. DDAH is a member of the amidinotransferase superfamily, and has conserved active–site residues including cysteine, histidine, and glutamate/aspartate that are integral to catalysis. In DDAH from Pseudomonas aeruginosa, the active–site Cys249 is activated as a nucleophile upon binding substrate, and forms a covalent intermediate concomitant with loss of the alkylamine leaving group. The active–site His162 has a dual role, first as a general acid in protonating the alkylamine leaving group and second as a general base in generating a hydroxide for attack on the covalent intermediate. The active–site Glu114 is essential for properly orienting and ionizing His162. The use of a substrate analog, S–methyl-L-thiocitrulline (SMTC), enabled development of a new method of continuously monitoring DDAH activity, allowing facile screening of inhibitors. Using this method, a haloacetamidine was identified as an active–site directed inactivator motif for DDAH, and other members of the amidinotransferase superfamily.

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