Browsing by Subject "Succinylarginine dihydrolase"
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Item Studies on the mechanism and inhibition of enzymes in the pentein superfamily(2012-05) Linsky, Thomas W.; Fast, Walter L.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.Item The design of halopyridine-based activity-based probes and mechanistic studies of succinylarginine dihydrolase(2015-05) Er, Joyce Ai Vee; Fast, Walter L.; Whitman, Christian P; Lee, Seongmin; Liu, Hung-wen; Iverson, Brent LAn important design aspect of covalent inactivators is the balance between reactivity, or reversibility of reaction, with nucleophiles in solution and reactivity with nucleophiles at a targeted protein site. We previously identified 4-halopyridines as fragment-sized covalent inactivators of the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Binding of these inactivators stabilizes the more reactive pyridinium form while the less reactive neutral form predominates in solution. Herein, we demonstrate that simple 2- and 4-chloropyridines are extensible as covalent modifiers of other proteins within the E. coli proteome, that halogen positioning can impart target selectivity, and that the targets include a subset of Cys-containing purine binding sites. As one specific example, inosine-5’-monophosphate dehydrogenase is shown to be labeled by a 2-chloropyridine at a catalytic Cys305 residue within the inosine binding site. These results indicate that a simple 2- or 4-chloropyridine core can have wider application as a warhead for incorporation into covalent inhibitors of proteins with diverse function. N-succinylarginine dihydrolase (AstB), like DDAH and arginine deiminase, is part of the amidinotransferase (AT) superfamily. AstB shows conservation of the catalytic residues and carries out a similar type of reaction as other hydrolases in the AT superfamily. Herein, we report the mechanistic studies of AstB and provide insights into how this enzyme performs its dihydrolase activity instead of a “mono” hydrolase reaction, which is more prevalent in this superfamily.