Nitric oxide (NO) is produced in different mammalian tissues by nitric oxide synthase (NOS), which has three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All NOS isoforms contain two domains, an oxygenase domain and a reductase domain. NO is an important transmitter of information between cells in many physiological processes; however, overproduction of this molecule may lead to health problems. Therefore, selective inhibition of NOS isoforms has useful therapeutic potential for treatment of certain diseases that can appear because of the pathological overproduction of nitric oxide. Producing useful isoform selective-inhibitors that bind to the active site in the oxygenase domain has proven to be difficult when based solely on the structure of these enzymes. Biophysical studies in combination with structural properties should provide better insights into isoform-specific inhibitor development. The first step of this study was to produce and purify truncated versions of NOS isozymes consisting of the oxygenase domain as they contain the active site of the enzyme. As a result of differences between humans and other mammals in the amino acids found in the second and third shells/layers surrounding the active site, all the experiments were performed with genes coding for human proteins. The major result of this project was the development of an Escherichia coli (E. coli) expression system to produce large amounts of pure protein. This system will allow for the testing of inhibitors that bind to the active site of NOS enzymes.

Nitric oxide (NO) is produced in different mammalian tissues by nitric oxide synthase (NOS), which has three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All NOS isoforms contain two domains, an oxygenase domain and a reductase domain. NO is an important transmitter of information between cells in many physiological processes; however, overproduction of this molecule may lead to health problems. Therefore, selective inhibition of NOS isoforms has useful therapeutic potential for treatment of certain diseases that can appear because of the pathological overproduction of nitric oxide. Producing useful isoform selective-inhibitors that bind to the active site in the oxygenase domain has proven to be difficult when based solely on the structure of these enzymes. Biophysical studies in combination with structural properties should provide better insights into isoform-specific inhibitor development. The first step of this study was to produce and purify truncated versions of NOS isozymes consisting of the oxygenase domain as they contain the active site of the enzyme. As a result of differences between humans and other mammals in the amino acids found in the second and third shells/layers surrounding the active site, all the experiments were performed with genes coding for human proteins. The major result of this project was the development of an Escherichia coli (E. coli) expression system to produce large amounts of pure protein. This system will allow for the testing of inhibitors that bind to the active site of NOS enzymes.