The overall goal of this proposal is to provide a molecular understanding of the structure/function relationships and enzymic mechanism of endothelial-type nitric oxide synthase (eNOS). The mechanistic hypothesis to be tested is based on a 3/2 coupling model between the reductase and the P450 oxidase mediated by CaM/Ca+2. We propose that CaM fixes a specific orientation between the FMN and heme centers without a drastic change in their physical distance. We also hypothesize that tetrahydrobiopterin (H4B), in addition to its structural role, is involved in the redox steps of oxygenase catalysis. We further suggest that the intimate spatial relationship of the L-arginine, H4B and heme sites leads to mutual regulation of ligand binding to each site. These specific interactions are proposed to be different in the three NOS isoforms. Such differences, together with the low similarity in the CaM target in each NOS isoform, determine the rate-limiting steps and the observed turnover numbers in the individual isoforms. To test these hypotheses we propose to: (i). Prepare eNOS and the two individual domains with a full complement of redox centers and use stoichiometric and potentiometric titrations to characterize the relative and absolute redox potential of each redox center; (ii).Evaluate interactions among L.arginine, H4B and heme binding sites by spectroscopic and kinetic methods with combinations of natural ligands and analogs; (iii). Characterize the electron transfer sequence and kinetics in eNOS and its oxygenase and reductase domains, and evaluate the proposed mechanism and the regulatory roles of CaM and H4B using rapid scan stopped-flow, rapid-freezing EPR and rapid-quenching/HPLC analyses to monitor individual redox centers. The planned studies will yield integrated knowledge about how eNOS (and other NOS isoforms) function, and provide structural information useful for the design of selective pharmacological inhibitors to controlling pathophysiological events associated with NO.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM056818-01A2
Application #
2841065
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1999-08-01
Project End
2003-07-31
Budget Start
1999-08-01
Budget End
2000-07-31
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
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Berka, Vladimir; Wang, Lee-Ho; Tsai, Ah-Lim (2008) Oxygen-induced radical intermediates in the nNOS oxygenase domain regulated by L-arginine, tetrahydrobiopterin, and thiol. Biochemistry 47:405-20
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Berka, Vladimir; Wu, Gang; Yeh, Hui-Chun et al. (2004) Three different oxygen-induced radical species in endothelial nitric-oxide synthase oxygenase domain under regulation by L-arginine and tetrahydrobiopterin. J Biol Chem 279:32243-51
Du, Mei; Yeh, Hui-Chun; Berka, Vladimir et al. (2003) Redox properties of human endothelial nitric-oxide synthase oxygenase and reductase domains purified from yeast expression system. J Biol Chem 278:6002-11

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