Nitric oxide (NO) is a key physiological messenger, but an excessive production of this molecule can be detrimental, leading to the onset or worsening of many pathological conditions. Dimethylarginine dimethylaminohydrolase (DDAH) is a key enzyme in the NO pathway, involved in the metabolism of asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA), which are both endogenous inhibitors of NO synthesis. Two isoforms of DDAH have been identified in humans, namely DDAH-1 and DDAH-2. DDAH inhibition represents a promising strategy in the treatment of NO overproduction under pathological conditions without affecting the homeostatic role of this messenger. In this work I described the design and synthesis of 12 novel potential DDAH inhibitors together with the development of a new UPLC-MS based assay to measure the activity of HEK293T cell lysates overexpressing recombinant human DDAH-1 in metabolizing ADMA into dimethylamine and L-citrulline. The same assay was used to assess the potential of the novel compounds, as well as of the well-known DDAH inhibitor L-257, to inhibit DDAH-1 catalyzed L-citrulline formation from ADMA. Three of the novel molecules (compounds 10a, 14a and 14b) showed very interesting inhibitory activity: in particular, the methylacylsulfonamide analogue of L-257 (10a) resulted in 13-fold higher inhibition potency than L-257 itself (98% of inhibition at 1mM, IC50 = 3±3 μM and Ki = 1±0 μM). This molecule was chosen for molecular dynamics simulations to study the putative mechanism for 10a inhibition of DDAH-1 activity. Furthermore, DDAH-1 and DDAH-2 were engineered introducing a FLAG-tag at the C-terminal of the proteins to allow their purification from the lysate components by immunoprecipitation. Although the purification protocol requires some further improvement, the fusion proteins did not show to be functionally affected by the modification.

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During catalysis the flavoprotein domain of neuronal nitric-oxide synthase (nNOS) shuttles NADPH-derived reducing equivalents from FAD to FMN and then to the P450-heme enabling heme-based oxygen activation and subsequent NO-synthesis. The binding of Ca�����-activated calmodulin (Ca�����-CaM) to nNOS alleviates inhibition of flavin mediated electron transfer within the diflavin domain, which is demonstrated by the increase in the rate of 2,6-dichioroindoiphenol (DCIP) reduction by 2 to 3 fold and that of cytochrome c����� by 10 to 20 fold. To investigate the effect of the Ca�����-CaM on the nNOS reductase activity, the steady-state kinetics of basal and CaM-stimulated reduction of these two substrates was studied. Parallel initial velocity patterns indicated that both substrates are reduced in a ping-pong mechanism. Product and dead-end inhibition data with DCIP as the electron acceptor were consistent with a di iso ping-pong bi-bi mechanism. In contrast, product and dead-end inhibition studies with cytochrome c����� as the second substrate were consistent with an iso (two-site) ping-pong mechanism. Ca�����-CaM did not alter the proposed kinetic mechanisms; however, it did effect to varying degrees the (k[subscript cat]/K[subscript]m) for the various substrates. The pH-dependence of basal and CaM-stimulated reduction of DCIP revealed that ionizable groups involved in the binding of substrates and catalysis are not altered by Ca�����-CaM. However, the activated cofactor does influence catalytic rate constants and/or ionizable groups involved in cytochrome c����� reduction. nNOS was found to abstract the pro-R (A-side) hydrogen from NADPH. Primary deuterium isotope effects (NADP(D)) and solvent isotope effects (SKIE) suggests that of the two half reactions, the reductive half reaction involving NADPH oxidation limits the overall reaction rate, but that hydride transfer to FAD is not the slow step. A small value of [supercript D](V/K)[subscript NADPH] (1.2-1.6) suggests hydride transfer is not the rate-limiting step within the reductive half-reaction. Large solvent kinetic isotope effects (SKIE) were observed on (V/K)[subscript cytc] for basal and CaM stimulated reduction of cytochrome c����� suggesting that proton uptake from the solvent limits the rate of the oxidative half-reaction. A small SKIE on V and (V/K)[subscript NADPH] indicates that proton uptake does not limit the overall reaction rate. Proton inventory analysis revealed multiple transition-state protons contributed to the observed SKIE. / Graduation date: 2001

Thesis (D. Med. Sci.)--University of Tartu, 2004. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xi, 174 p. : ill. (some col.). Advisor: Anthony Young, Molecular, Cellular, and Developmental Biology Program. Includes bibliographical references (p. 154-174).

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains x, 156 p.; also includes graphics (some col.) Includes bibliographical references (p. 146-156). Available online via OhioLINK's ETD Center

Tetrahydrobiopterin (BH4) is a limiting cofactor for nitric oxide synthase (NOS) catalysed conversion of L-arginine to nitric oxide and citrulline. Content of BH4 in mammalian cells is regulated at many levels, but most important is de novo biosynthesis from GTP. GTP cyclohydrolase (GTPCH) is the rate-limiting enzyme for the de novo synthesis of BH4. While various immunostimulants, hormones and growth factors have been reported to increase GTPCH mRNA levels and intracellular biopterin (BH4 degradation product), it is not known whether these factors act at the level of GTPCH gene transcription. To test this I utilised 1, 3 and 6 kb 5'upstream GTPCH gene sequence in a secreted alkaline phosphatase reporter vector (SEAP). These constructs were stably transfected in PC-12 cells and rat aortic smooth muscle cells, and the cells were treated with various immunostimulants and growth factors in order to determine whether these factors could enhance GTPCH gene transcription. Intracellular biopterin levels were also measured to confirm that the upregulation of the SEAP-reporter correlated with a rise in biopterin. Our investigations conclude that transcriptional regulation of the GTPCH gene is indeed a major site for control of intracellular BH4 levels. In further experiments, we have characterised the binding of [3H]BH4 to endothelial NOS (eNOS) and examined influences of the substrate, arginine, on the BH4 binding. In addition we selected tetrahydropterins (that support NOS catalysis) and dihydropterins (that are catalytically incompetent) to determine the extent to which modifications of BH4 alter pterin binding affinity to eNOS. Dihydropterins are unable to support NOS catalysis. Studies showed for the first time that dihydropterins, but not tetrahydropterins, support superoxide generation by eNOS. We also have determined that eNOS may be able to produce NO in the absence of BH4 cofactor from the reaction intermediate hydroxyarginine. We have characterised this reaction and are able to provide a plausible mechanism for the NOx generation from eNOS in the absence of BH4 cofactor.

A limitation to investigations of homodimeric protein dissociation is that the signals produced from methods such as fluorescence and circular dichroism represent both dissociation and protein unfolding that may be occurring simultaneously within a sample. Although size exclusion chromatography examines the state of a protein’s quaternary structure, complicated overlapping peaks representative of oligomer and monomer can result. To address these limitations the mixed dimer system has been adopted to investigate the dissociation of a homodimeric protein. A mixed dimer is a species in which each subunit of a homodimeric protein is associated with a different affinity tag. The two tags used are the His6-tag and the Glu7-tag. Such a mixed dimer will bind to a metal chelating column such as Ni-NTA so long as the dimer is intact. Denaturant- or temperature induced dimer dissociation can be detected by the amount of Glu7-tagged subunit present in the unbound fraction after the protein is passed over an Ni-NTA resin. SDS PAGE and densitometry assess the amount of Glu7-tagged subunit present in those unbound fractions. The experimental conditions necessary to implement this method were developed, and then applied to mammalian inducible nitric oxide synthase (iNOS) and Staphylococcus aureus NOS (SaNOS). With respect to both urea and temperature, the stability of SaNOS is higher than that of iNOS in spite of the bacterial enzyme having a much smaller dimer contact surface. We have also used the mixed dimer method to estimate an equilibrium dissociation constant (KD) for iNOS dissociation of no greater than 2.3μM. This value is compared to the results obtained for iNOS by analytical ultracentrifugation, which can characterize protein complexes and their stoichiometry. / Thesis (Master, Chemistry) -- Queen's University, 2007-10-26 15:28:14.182

Thesis (Ph. D.)--University of Lund, 1998. / Originally issued as the author's doctoral thesis. Includes bibliographical references.