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Rob Whitehouse, Cleveland Clinic
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Scientists Solve Active Site of Structure of Enzyme that Produces Nitric Oxide; Discovery Suggests Possible New Ways to Design Novel Drugs for Several Human Diseases

La Jolla, California. October 17 -- Scientists at The Skaggs Institute for Chemical Biology and the Department of Molecular Biology at The Scripps Research Institute, led by Drs. John Tainer and Elizabeth Getzoff, in collaboration with a team led by Dr. Dennis Stuehr at the Cleveland Clinic, have solved the structure of the active site of the enzyme that regulates the activity of nitric oxide, or NO. Since NO is an unconventional biological signal whose activities range from blood pressure regulation to antimicrobial defense to nervous system information and memory, understanding the structure of the enzyme that produces it is crucial to designing drugs to turn NO on and off. Scientists predict that NO inhibitors may be used to treat such diseases as high blood pressure, septic shock, stroke, cancer, and impotence. Given its role in neurotransmission, NO may have an effect on treating memory disorders and learning.

The paper, "The Structure of Nitric Oxide Synthase Oxygenase Domain and Inhibitor Complexes," by Drs. Brian R. Crane, Andrew S. Arvai, Ratan Gachhui, Chaoqun Wu, Dipak K. Ghosh, Elizabeth D. Getzoff, Dennis J. Stuehr, and John A. Tainer, appears in today's issue of Science.

According to Tainer, "Having this structure is the difference between working blind and seeing what you're doing in terms of understanding and drug design."

The structure of this key portion of nitric oxide synthase (NOS) helps researchers understand not only how NO is produced in the body but also how NO production is controlled. Nitric oxide is a small, short-lived, inorganic molecule that functions in mammals as an essential chemical messenger for many physiological processes and as a protective poison against pathogens and cancer. At low concentrations it acts as a signal to control blood pressure, prevent blood clotting, transmit nerve impulses in contractile and sensory tissues, process sensory input, form memories, and allow learning.

In contrast, the immune system produces high concentrations of NO and exploits its reactive properties to combat bacteria, intracellular parasites, viruses and tumor cells. Due to its unstable and membrane diffusible nature, NO differs from other neurotransmitters and hormones in that it is not regulated by storage, release or targeted degradation, but rather solely by synthesis.

Because NO acts as a signal in low amounts and a toxin in high amounts, its production is carefully balanced in healthy humans depending on the state of the organism. Pathologies thought to involve too little NO production include hypertension, impotence, arteriosclerosis, and a susceptibility to infection. Diseases linked to excessive NO production include immune-type diabetes, neurotoxicity associated with aneurysm, stroke and reperfusion injury, inflammatory bowel disease, rheumatoid arthritis, cancer, septic shock, multiple sclerosis and transplant rejection.

According to Dr. Solomon Snyder, a neuroscientist at Johns Hopkins University whose research group was the first to clone and sequence NOS, "NO appears to be one of the most important messenger molecules in the body. Excess production appears to cause brain damage from stroke and also inflammatory conditions. Drugs that block the enzyme could be important therapeutically; this breakthrough may allow scientists to begin to design drugs to inhibit it." The first three-dimensional structures of the catalytic site of NOS show in atomic detail how the enzyme recognizes the amino acid arginine, its substrate, and oxidizes it to form the biological signal NO. Stuehr stated, "We now have a clear understanding of where the reactive groups are located and how the enzyme can control their interaction." The researchers believe that the unexpected discovery of two adjacent binding sites for the NOS inhibitor imidazole in the active site promises to aid in the design of drugs to modulate NOS activity and prevent NO overproduction.

Dual-function inhibitors that simultaneously bind both of these sites would block both arginine and oxygen binding, creating an expanded dual-site binding region to increase affinity and prevent the formation of toxic, reactive oxygen species. Since the characteristics of NOS inhibition vary among different NOS types, these dual-function inhibitors also may lead to new drugs that target only one of the various forms of NOS, thereby limiting potential side effects.

The chemistry NOS uses to produce NO is complicated and unique in biology, and its structure is completely different from other oxygenase enzymes involved in hormone synthesis and the detoxification of harmful compounds. However, a comparison provides insight into the aspects of these enzymes that are key for the similarities and differences in the reactions they catalyze. According to Tainer, this should aid researchers in reproducing these biological reactions in the laboratory for the design of drugs or other desirable compounds.

A technique known as protein crystallography was used to determine the NOS structures. This involves diffracting x-rays off of crystals grown from the highly purified enzyme. The x-ray diffraction experiment provides all the information necessary to create an atomic image of the protein. X-ray radiation was needed in this experiment because the diffraction only occurs when the size of the object is similar to the wavelength size of the radiation.

Funding for the study was obtained from the Skaggs Institute for Research and the National Institutes of Health, the latter of which accounted for approximately 15% of the resources.

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