Studies on the mechanism and inhibition of enzymes in the pentein superfamily

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.