La Jolla, California. October 17 -- Scientists at The Skaggs Institute forChemical Biology and the Department of Molecular Biology at The Scripps ResearchInstitute, led by Drs. John Tainer and Elizabeth Getzoff, in collaboration witha team led by Dr. Dennis Stuehr at the Cleveland Clinic, have solved thestructure of the active site of the enzyme that regulates the activity of nitricoxide, or NO.
Since NO is an unconventional biological signal whose activitiesrange from blood pressure regulation to antimicrobial defense to nervous systeminformation and memory, understanding the structure of the enzyme that producesit is crucial to designing drugs to turn NO on and off. Scientists predict thatNO inhibitors may be used to treat such diseases as high blood pressure, septicshock, stroke, cancer, and impotence. Given its role in neurotransmission, NOmay have an effect on treating memory disorders and learning.
The paper, "The Structure of Nitric Oxide Synthase Oxygenase Domain andInhibitor 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 betweenworking blind and seeing what you're doing in terms of understanding and drugdesign."
The structure of this key portion of nitric oxide synthase (NOS) helpsresearchers understand not only how NO is produced in the body but also how NOproduction is controlled. Nitric oxide is a small, short-lived, inorganicmolecule that functions in mammals as an essential chemical messenger for manyphysiological processes and as a protective poison against pathogens and cancer.At low concentrations it acts as a signal to control blood pressure, preventblood 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 andexploits its reactive properties to combat bacteria, intracellular parasites,viruses and tumor cells. Due to its unstable and membrane diffusible nature, NOdiffers from other neurotransmitters and hormones in that it is not regulated bystorage, 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 ofthe organism. Pathologies thought to involve too little NO production includehypertension, 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, multiplesclerosis and transplant rejection.
According to Dr. Solomon Snyder, a neuroscientist at Johns HopkinsUniversity whose research group was the first to clone and sequence NOS, "NOappears to be one of the most important messenger molecules in the body. Excessproduction appears to cause brain damage from stroke and also inflammatoryconditions. Drugs that block the enzyme could be important therapeutically; thisbreakthrough may allow scientists to begin to design drugs to inhibit it."
The first three-dimensional structures of the catalytic site of NOS showin atomic detail how the enzyme recognizes the amino acid arginine, itssubstrate, and oxidizes it to form the biological signal NO. Stuehr stated, "Wenow have a clear understanding of where the reactive groups are located and howthe enzyme can control their interaction." The researchers believe that theunexpected discovery of two adjacent binding sites for the NOS inhibitorimidazole in the active site promises to aid in the design of drugs to modulateNOS activity and prevent NO overproduction.
Dual-function inhibitors that simultaneously bind both of these siteswould block both arginine and oxygen binding, creating an expanded dual-sitebinding region to increase affinity and prevent the formation of toxic, reactiveoxygen species. Since the characteristics of NOS inhibition vary among differentNOS types, these dual-function inhibitors also may lead to new drugs that targetonly one of the various forms of NOS, thereby limiting potential side effects.
The chemistry NOS uses to produce NO is complicated and unique inbiology, and its structure is completely different from other oxygenase enzymesinvolved in hormone synthesis and the detoxification of harmful compounds.However, a comparison provides insight into the aspects of these enzymes thatare key for the similarities and differences in the reactions they catalyze.According to Tainer, this should aid researchers in reproducing these biologicalreactions in the laboratory for the design of drugs or other desirablecompounds.
A technique known as protein crystallography was used to determine theNOS structures. This involves diffracting x-rays off of crystals grown from thehighly purified enzyme. The x-ray diffraction experiment provides all theinformation necessary to create an atomic image of the protein. X-ray radiationwas needed in this experiment because the diffraction only occurs when the sizeof the object is similar to the wavelength size of the radiation.
Funding for the study was obtained from the Skaggs Institute forResearch and the National Institutes of Health, the latter of which accountedfor approximately 15% of the resources.
Materials provided by Scripps Research Institute. Note: Content may be edited for style and length.
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