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Title: Oxygen activation during neuronal NOS reaction
Author: Papale, Davide
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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Nitric oxide synthase (NOS) catalyzes nitric oxide (NO) production in a two step reaction. The first step involves L-arginine being oxidised to NG-hydroxy-L-arginine (NOHA) that remains bound to the enzyme before it is oxidised to NO and L-citrulline in the second step. The three mammalian isozymes are homodimeric enzymes and each subunit is composed of a reductase and an oxygenase domain. The oxygenase domain contains the arginine binding site, one cysteine ligated heme thiolate, and one H4B molecule ((6R)-5,6,7,8-Tetrahydrobiopterin). The enzyme’s substrates are O2, L-arginine (or NOHA) and NADPH. H4B is an essential cofactor for NO production by NOS and its roles are in dimerization and as redox cofactor. The mechanism of the reaction between L-arg and oxygen in the active site is currently uncertain. Therefore in order to alter the course of the reaction site-directed mutagenesis was conducted: Glycine 586 of nNOS was replaced by a serine residue (G586S nNOSoxy) and expressed and purified from E.coli. Stopped flow kinetic experiments showed the formation of a novel reaction intermediate during G586S nNOSoxy catalysis in the presence of H4B and substrate, subsequent to the formation of the oxy-ferrous compound. It is suggested that the new intermediate is formed as a transient along the path of the NO production reaction and has spectroscopic resemblance to the putative P450 active species, the oxy-ferryl compound. Crystals of the G586S mutant have been obtained and the solved x-ray structure shows the newly introduced serine residue pointing toward the guanidinium group of L-arg, reinforcing its involvement in the stabilization of a reaction intermediate.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available