Platinum(II) olefin hydroarylation catalysts: tuning selectivity for the anti-Markovnikov product Tandem catalytic ester hydrogenation and deoxygenation of polyethylene terephthalate

Thesis (Master's)--University of Washington, 2016-06

Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-α

Importin-alpha is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-alpha, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-alpha and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX10-12KRRK) that can be used to identify novel nuclear proteins.

Preparation and catalytic properties of ZrO2-Al2O3 composite oxide supported nickel catalysts for methane reforming with carbon dioxide

ZrO2-Al2O3 composite oxides and supported Ni catalysts were prepared, and characterized by N-2 adsorption/desorption, X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) techniques. The catalytic performance and carbon deposition was also investigated. This mesoporous composite oxide is shown to be a promising catalyst support. An increase in the catalytic activity and stability of methane and carbon dioxide reforming reaction was resulted from the zirconia addition, especially at 5wt% ZrO2 content. The Ni catalyst supported ZrO2-Al2O3 has a strong resistance to sintering and the carbon deposition in a relatively long-term reaction.

Molecular modelling of human CYP1B1 substrate interactions and investigation of allelic variant effects on metabolism

Molecular modelling of human CYP1B1 based on homology with the mammalian P450, CYP2C5, of known three-dimensional structure is reported. The enzyme model has been used to investigate the likely mode of binding for selected CYP1B1 substrates, particularly with regard to the possible effects of allelic variants of CYP1B1 on metabolism. In general, it appears that the CYP1B1 model is consistent with known substrate selectivity for the enzyme, and the sites of metabolism can be rationalized in terms of specific contacts with key amino acid residues within the CYP1B1 heme locus. Further-more, a mode of binding interaction for the inhibitor, a-naphthoflavone, is presented which accords with currently available information. The current paper shows that a combination of molecular modelling and experimental determinations on the substrate metabolism for CYP1B1 allelic variants can aid in the understanding of structure-function relationships within P450 enzymes. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

A novel laponite clay-based Fe nanocomposite and its photo-catalytic activity in photo-assisted degradation of Orange II

A novel laponite RD clay-based Fe nanocomposite (Fe-Lap-RD) has been successfully synthesized through a reaction between a solution of iron salt and an aqueous dispersion of laponite RD clay. The X-ray diffraction (XRD) results reveal that the Fe-Lap-RD mainly consists of Fe2O3 (maghemite) and Fe2Si4O10(OH)2 (iron silicate hydroxide), which have tetragonal and monoclinic structures, respectively, and has a high specific surface area as well as a high pore volume. The photo-catalytic activity of the Fe-Lap-RD was examined in the photo-assisted degradation of an organic azo dye Orange II. It was found that the mineralization of Orange 11 undergoes a slower kinetics than discoloration, and 70% total organic carbon (TOC) of 0.2 mM Orange 11 can be removed in 90 min, implying that the Fe-Lap-RD exhibited a high photo-catalytic activity in the presence of H2O2 and UV light (254 nm) in the photo-assisted degradation of Orange II. In addition, our experiments also illustrate that the Fe-Lap-RD has a long-term stability but is of low cost. This study illustrates the possibility of photo-assisted degradation of organic compounds without the requirements to remove the Fe ions after reaction. Two possible catalytic reaction mechanisms are also proposed. (C) 2003 Elsevier Science Ltd. All rights reserved.

Catalytic properties of heteropolyacids supported on MCM-41 mesoporous silica for hydrocarbon cracting reactions

It is known that MCM-41 structures have very weak acid sites because of the lack of the bridging hydroxyl groups present in zeolites. Strong acidity however is required for the potential use of these materials in some specific applications such as: cracking and hydrotreating of heavy residue molecules, cracking of waste plastic, etc. The acidity enhancement of the MCM-41 materials was assessed using the n-hexane and polyethylene cracking reactions. MCM-41 samples were impregnated using heteropolyacid (HPA) such as tungestophospheric acid. The catalyst samples were characterized also by x-ray diffraction and benzene adsorption.

Association of cone thermogenesis and volatiles with pollinator specificity in Macrozamia cycads

Cone traits (volatile components and thermogenesis) of three cycad species in the genus Macrozamia were examined for differences related to their specific insect pollinators, the weevil, Tranes spp., or the thrips, Cycadothrips chadwicki. Linalool (>80% of emissions) dominated cone volatile components of M. machinii (Tranes-pollinated) and beta-myrcene was a minor component (

The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-Angstrom crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 Angstrom, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.

The critical role of tryptophan-116 in the catalytic cycle of dimethylsulfoxide reductase from Rhodobacter capsulatus

In dimethylsulfoxide reductase of Rhodobacter capsulatus tryptophan-116 forms a hydrogen bond with a single oxo ligand bound to the molybdenum ion. Mutation of this residue to phenylalanine affected the UV/visible spectrum of the purified Mo-VI form of dimethylsulfoxide reductase resulting in the loss of the characteristic transition at 720 nm. Results of steady-state kinetic analysis and electrochemical studies suggest that tryptophan 116 plays a critical role in stabilizing the hexacoordinate monooxo Mo-VI form of the enzyme and prevents the formation of a dioxo pentacoordinate Mo-VI species, generated as a consequence of the dissociation of one of the dithiolene ligands of the molybdopterin cofactor from the Mo ion. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

A numerical approach to modeling the catalytic voltammetry of surface-confined redox enzymes

A finite difference method for simulating voltammograms of electrochemically driven enzyme catalysis is presented. The method enables any enzyme mechanism to be simulated. The finite difference equations can be represented as a matrix equation containing a nonlinear sparse matrix. This equation has been solved using the software package Mathematica. Our focus is on the use of cyclic voltammetry since this is the most commonly employed electrochemical method used to elucidate mechanisms. The use of cyclic voltammetry to obtain data from systems obeying Michaelis-Menten kinetics is discussed, and we then verify our observations on the Michaelis-Menten system using the finite difference simulation. Finally, we demonstrate how the method can be used to obtain mechanistic information on a real redox enzyme system, the complex bacterial molybdoenzyme xanthine dehydrogenase.

Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane

The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.

Assessment of arginine 97 and lysine 72 as determinants of substrate specificity in cytochrome P450 2C9 (CYP2C9)

CYP2C9 is distinguished by a preference for substrates bearing a negative charge at physiological pH. Previous studies have suggested that CYP2C9 residues R97 and K72 may play roles in determining preference for anionic substrates by interaction at the active site or in the access channel. The aim of the present study was to assess the role of these two residues in determining substrate selectivity. R97 and K72 were substituted with negative, uncharged polar and hydrophobic residues using a degenerate polymerase chain reaction-directed strategy. Wild-type and mutant enzymes were expressed in bicistronic format with human cytochrome P450 reductase in Escherichia coli. Mutation of R97 led to a loss of holoenzyme expression for R97A, R97V, R97L, R97T, and R97E mutants. Low levels of hemoprotein were detected for R97Q, R97K, R97I, and R97P mutants. Significant apoenzyme was observed, suggesting that heme insertion or protein stability was compromised in R97 mutants. These observations are consistent with a structural role for R97 in addition to any role in substrate binding. By contrast, all K72 mutants examined (K72E, K72Q, K72V, and K72L) could be expressed as hemoprotein at levels comparable to wild-type. Type I binding spectra were obtained with wildtype and K72 mutants using diclofenac and ibuprofen. Mutation of K72 had little or no effect on the interaction with these substrates, arguing against a critical role in determining substrate specificity. Thus, neither residue appears to play a role in determining substrate specificity, but a structural role for R97 can be proposed consistent with recently published crystallographic data for CYP2C9 and CYP2C5.

Comparison of specificity of quantitative myocardial contrast echocardiography for diagnosis of coronary artery disease in patients with versus without diabetes mellitus

Impaired coronary flow reserve is widely reported in diabetes mellitus (DM) but its effect on myocardial contrast echocardiography (MCE) is unclear. We sought to identify whether DM influences the accuracy of qualitative and quantitative assessment of coronary artery disease (CAD) using MCE in 83 patients who underwent coronary angiography (60 men, 27 with DM; 56 +/- 11 years;). Destruction replenishment imaging was performed at rest and after combined dipyridamole-exercise stress testing. Ischemia was identified by the development of new wall motion abnormalities, qualitative MCE (new perfusion defects apparent 1 second after flash during hyperemia), and quantitative MCE (myocardial blood flow reserve < 2.0 in the anterior circulation). Qualitative and quantitative assessment of perfusion was feasible in 100% and 92% of patients, respectively. Significant left anterior descending coronary stenosis (> 50% by quantitative angiography) was present in 28 patients (including 8 with DM); 55 patients had no CAD (including 19 with DM). The myocardial blood flow reserve was reduced in patients with coronary stenosis compared with those with no CAD (1.6 +/- 1.1 vs 3.8 +/- 2.5, p < 0.001). Among patients with no CAD, those with DM had an impaired flow reserve compared with control patients without DM (2.4 +/- 1.0 vs 4.5 +/- 2.8, p = 0.003). In conclusion, DM significantly influenced the quantitative, but not the qualitative, assessment of MCE, with a marked reduction in specificity in patients with DM. (c) 2005 Elsevier Inc. All rights reserved.

Human tartrate-resistant acid phosphatase becomes an effective ATPase upon proteolytic activation

Proteolytic, cleavage in an exposed loop of human tartrate-resistant acid phosphatase (TRAcP) with trypsin leads to a significant increase in activity. At each pH value between 3.25 and 8.0 the cleaved enzyme is more active. Substrate specificity is also influenced by proteolysis. Only the cleaved form is able to hydrolyze unactivated substrates efficiently, and at pH > 6 cleaved TRAcP acquires a marked preference for ATP. The cleaved enzyme also has altered sensitivity to inhibitors. Interestingly, the magnitude and mode of inhibition by fluoride depends not only on the proteolytic state but also pH. The combined kinetic data imply a role of the loop residue D158 in catalysis in the cleaved enzyme. Notably, at low pH this residue may act as a proton donor for the leaving group. In this respect the mechanism of cleaved TRAcP resembles that of sweet potato purple acid phosphatase. (c) 2005 Elsevier Inc. Ail rights reserved.

Molecular basis of intramolecular electron transfer in sulfite-oxidizing enzymes is revealed by high resolution structure of a heterodimeric complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit

Sulfite-oxidizing molybdoenzymes convert the highly reactive and therefore toxic sulfite to sulfate and have been identified in insects, animals, plants, and bacteria. Although the well studied enzymes from higher animals serve to detoxify sulfite that arises from the catabolism of sulfur-containing amino acids, the bacterial enzymes have a central role in converting sulfite formed during dissimilatory oxidation of reduced sulfur compounds. Here we describe the structure of the Starkeya novella sulfite dehydrogenase, a heterodimeric complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit, that reveals the molecular mechanism of intramolecular electron transfer in sulfite-oxidizing enzymes. The close approach of the two redox centers in the protein complex (Mo-Fe distance 16.6 angstrom) allows for rapid electron transfer via tunnelling or aided by the protein environment. The high resolution structure of the complex has allowed the identification of potential through-bond pathways for electron transfer including a direct link via Arg-55A and/or an aromatic-mediated pathway. A potential site of electron transfer to an external acceptor cytochrome c was also identified on the SorB subunit on the opposite side to the interaction with the catalytic SorA subunit.