Over the last several years nitric oxide (-NO) has emerged as an important mediator in several key physiological processes including blood vessel homeostasis, neuronal communication, and host response to infection. The enzymology and regulation of -NO biosynthesis hold the keys to the complete understanding and ultimate control over the processes regulated by -NO. Studies in this area will provide important new information concerning blood pressure control and cell-mediated killing. -NO appears to play a key role in stasis and killing of intracellular parasites. In addition, sustained and elevated generation of -NO can be deleterious and may be important in the etiology of endotoxic shock, inflammation-related tissue damage, neurotoxicity, carcinogenesis. The overall long-term goal for this project is a thorough characterization of the mechanism of the enzyme -NO synthase in order to develop the ability to control -NO synthesis through the rational design of agents based on this mechanistic understanding. The mechanism of the oxidation of L-arginine to nitric oxide (-NO) and citrulline by -NO synthase (EC 1. 14.23) is poorly understood. Except for the fact that N-hydroxylation is the first step in the reaction sequence, virtually nothing is known about the subsequent chemical and/or enzymatic steps. Intermediates beyond the N-hydroxylation step have yet to be elucidated although several have been proposed. The general experimental approach will involve continued mechanistic studies including (a) the function of prosthetic groups (b)the structural requirements of the binding site for the substrate as well the design of arginine analogs as mechanistic tools to unravel the oxidative chemistry occurring at the guanido nitrogen, and (c) the design of isoform selective inhibitors of the -NO synthase to probe in vivo function and point the way toward the development of new therapeutic agents.
The specific aims are as follows: (1) mechanistic studies of murine macrophage -NO synthase including (i) function of heme and reduced pterin requirement, (ii) NADPH utilization, (iii) role of the flavin cofactors (iv) use of arginine analogs as probes of mechanism; (2) further characterization of the inducible -NO synthase including biophysical studies ; (3) synthesis and characterization of the action of mechanism- based inhibitors of the -NO synthase ; (4) over-expression of the -NO synthase from the murine macrophage and rat cerebellum; (5) selected site-directed mutagenesis of the macrophage -NO synthase to further probe mechanism; and (6) characterization of the murine macrophage malic enzyme including (i) inducibility by cytokines and/or exogenous immunostimulants (ii) relationship to the -NO synthase.
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