Dimethylarginine dimethylaminohydrolase (DDAH) indirectly regulates nitric oxide production by hydrolyzing methylated arginines, which are endogenous nitric oxide synthase inhibitors. This enzyme is a member of the mechanistically diverse pentein superfamily, which contains hydrolase, dihydrolase, and amidinotransferase enzymes. These enzymes are proposed to use the same first catalytic step, followed by partitioning into their respective activities. Here, variants of DDAH that can catalyze the dihydrolase and amidinotransfer reactions are presented, as well as a variant of succinylarginine dihydrolase which catalyzes a single hydrolysis reaction. The results experimentally demonstrate that the proposed common catalytic intermediate can be used for several different reactions. The results suggest that enzymes in the pentein superfamily may have evolved divergently from a catalytically promiscuous ancestor. The control DDAH asserts over nitric oxide production makes it an attractive drug target for disease states marked by pathological overproduction of nitric oxide. Only a limited number of inhibitors different from substrate are reported, due in part to lack of robust assays for high-throughput screening of compound libraries. Therefore, high-throughput assays were developed, optimized, and validated to screen for inhibitors of Pseudomonas aeruginosa DDAH and human DDAH-1. These assays were used to screen three commercial libraries totaling 6,466 compounds. One drug in phase III clinical trials, ebselen, was identified and characterized as a bioavailable, rapid covalent inactivator of DDAH both in vitro and in cultured cells. Four "fragment-sized" inhibitors were also identified and characterized in the screening, including 4-halopyridines and benzimidazole-like compounds. The 4-halopyridines, not previously known to modify proteins, act as quiescent affinity labels to selectively inactivate DDAH, and the benzimidazole-like compounds are competitive, rapidly reversible inhibitors of DDAH. These diverse molecules serve as starting points for the development of molecular probes and therapeutic drugs to reduce pathological overproduction of nitric oxide. / text

Nitric oxide (NO) overproduction is correlated with numerous human diseases, such as arthritis, asthma, diabetes, inflammation and septic shock. The enzyme activities of both NO synthase (NOS) and dimethylarginine dimethylaminohydrolase-1 (DDAH-1) promote NO production. DDAH-1 mainly colocalizes in the same tissues as the neuronal isoform of NOS and catabolizes the endogenously-produced competitive inhibitors of NOS, N[omega]-monomethyl-L-arginine (NMMA) and asymmetric N[omega], N[omega]-dimethyl-L-arginine (ADMA). Inhibition of DDAH-1 leads to elevated concentrations of NMMA and ADMA, which subsequently inhibit NOS. To better understand DDAH-1, I first characterized the catalytic mechanism of human DDAH-1, where Cys274, His173, Asp79 and Asp127 form a catalytic center. Particularly, Cys274 is an active site nucleophile and His173 plays a dual role in acid/base catalysis. I also studied an unusual mechanism for covalent inhibition of DDAH-1 by S-nitroso-L-homocysteine (HcyNO), where an N-thiosulfoximide adduct is formed at Cys274. Using a combination of site directed mutagenesis and mass spectrometry, we found that many residues that participate in catalysis also participate in HcyNO mediated inactivation. Following these studies, I then screened a small set of known NOS inhibitors as potential inhibitors of DDAH-1. The most potent of these, an alkylamidine, was selected as a scaffold for homologation. Stepwise lengthening of the alkyl substituent changes an NOS-selective inhibitor into a dual-targeted NOS/DDAH-1 inhibitor then into a DDAH-1 selective inhibitor, as seen in the inhibition constants of N5-(1-iminoethyl)-, N5-(1-iminopropyl)-, N5-(1-iminopentyl)- and N5-(1-iminohexyl)-L-ornithine for neuronal NOS (1.7, 3, 20, >1,900 [mu]M, respectively) and DDAH-1 (990, 52, 7.5, 110 [mu]M, respectively). X-ray crystal structures suggest that this selectivity is likely due to active site size differences. To rank the inhibitors' in vivo potency, we constructed a click-chemistry based activity probe to detect inhibition of DDAH-1 in live mammalian cell culture. In vivo IC50 values for representative alkylamidine based inhibitors were measured in living HEK293T cells. Future application of this probe will address the regulation of DDAH-1 activity in pathophysiological states. In summary, this work identifies a versatile scaffold for developing DDAH targeted inhibitors to control NO overproduction and provides useful biochemical tools to better understand the etiology of endothelial dysfunction. / text