Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains xi, 124 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 81-87).

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains x, 210 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 154-185).

D. Tech. Chemistry. / Demonstrates the possibility of preparing large surface coatings of monodispersed spherical particles of chromium(III) oxide by using the ACG method for solar heating and magneto-optic technologies. t is aimed in the current study to investigate the photo-induced ESR phenomena in a-Cr2O3 mono-dispersed spherical particles using elimination of 160mW Nd:YAG laser of ˜1064 nm and a pulse repetition frequency of 30Hz, in the temperature range of 150 up to 315 K.

Variable temperature ESR studies of radicals generated photolytically from simple aliphatic alcohols and ethers in cyclopropane solution reveal complex linewidth effects. Isotropic modulation of the proton hyperfine splittings (hfs) through restricted rotation about C-0 and C-C single bonds is observable in the region 230>T>150K. Such effects can be distinguished from anisotropic viscosity-dependent line broadening. In spectra from alcohol radicals resolved 2nd order structure causes no ambiguity in the interpretation. Restricted rotation about Ca-0H modulates aaH and aBH out-of-phase with a0H in the series RCHOH [R= CH3, C2H5, C2H5CH2, (CH3)2CHCH2, (CH3)3CCH2]. A general model for the process is discussed. In cases three and four restricted Co-C rotation allows the diastereotopic inequivalence of the 6-protons to be manifested as a broadening of MB = 0 components. Preferred conformations consistent with all the foregoing modulation effects and with observed HB and HY splittings are presented. The analysis is supported by results for radicals RCHOR' from related ethers and by spectral simulation. The spectrum of the 1-hydroxycyclohexyl radical demonstrates previously unobserved fine structure and a low-temperature linewidth effect tentatively attributed to radical site inversion. A second series of alcohol-derived radicals R1R2R3CCHOH with an increasingly bulky Ca substituent has been studied. The Ha hfs provide evidence of a steric flattening not hitherto observed. This effect correlates well with literature values of steric parameters for the R1R2R3C substituent. In the case R1,R2 = CH3, R3 = C2H5 an observed specific y-H interaction is assigned to a locked conformation of the crowded system. A series of highly alkylated cyclic ethers has been examined. The dramatic temperature-dependent changes in the spectrum of the 5,5-dimethyl-l,3-dioxan-2-yl radical are attributed to restricted ring flipping. A fast exchange limit spectrum has been obtained for the first time in such systems, allowing evaluation of thermodynamic parameters. the 2,4,8,10-tetraoxyspiro[5,5]undecan-3-yl radical exhibits similar behaviour. The 2,2,5,5-tetramethyl and 5,5-diethyl-2,2-dimethyl-l,3--dioxan-4-yl radicals have fixed conformations which give rise to enhanced values of ayH in agreement with theoretical calculations. In the latter case a splitting of 4.27 G is assigned to a single y-methylene proton in behaviour analogous to R1R2R3CCHOH.

Sulfite oxidising enzymes are found in all forms of life and play an important role in detoxification of sulfite produced through biochemical processes. All known sulfite oxidising enzymes share a common molybdenum active site. The sulfite dehydrogenase (SDH) from the soil bacterium Starkeya novella differs from the vertebrate sulfite oxidases (SO) in that the heme and Mo subunits are tightly associated rather than connected by a flexible hinge. This structural integrity makes SDH an ideal model enzyme for the study of enzymatic sulfite oxidation without the complications of structural changes underlying catalysis. In human sulfite oxidase (HSO) the substitution of a conserved active site amino acid residue, Arg-160 for Gln, results in a lethal disease. A number of independent studies have been carried out in order to understand the effects of this substitution on catalysis in both human (HSO) and chicken sulfite oxidising enzymes (CSO). The focus of this work is the analogous residue in SDH, Arg 55. A number of active site substitutions have been investigated, including SDHR55Q, an analogous substitution to the lethal mutation identified in humans. In addition, the properties of the Arg residue have also been probed using a substitution to a hydrophobic residue, Met (SDHR55M) and a substitution to the positively charged Lys (SDHR55K). A fourth active site substitution, SDHH57A, was also investigated as the crystal structure of this variant indicated that His-57 plays a role in stabilising the position of Arg-55 in SDH. It was of interest to determine the effect of the instability in the position of Arg-55 on the catalytic parameters of the SDHH57A. The kinetic properties of the substituted enzymes were investigated using steady-state assays with cytochrome c as an electron acceptor. When the positive charge was lost in the case of SDHR55M and SDHR55Q, a dramatic increase in the KM (sulfite - app) of 2 – 3 orders of magnitude resulted. This indicates that the positive charge on Arg-55 is important for substrate binding. All the Arg-55 variants studied were found to have lower turnover numbers than the wild type, in particular, SDHR55Q was found to have a reduced kcat (108 s-1 vs 345 s-1 for SDHWT at pH 8). The changes in the Mo centre underlying the altered kinetic properties were investigated in detail using EPR spectroscopy of the intermediate MoV oxidation state in SDHR55Q and SDHH57A. Similar to what has been noted for HSOR160Q, a sulfate blocked form was observed at pH 6 using pulsed EPR experiments, suggesting that this substitution causes an inhibition of the hydrolysis step required to release the reaction product, sulfate. This could be a further reason for the poor catalytic activity of SDHR55Q, in particular, a reason for the low turnover rate of this variant. Unlike what was noted in HSOR160Q, where the substituted enzyme showed a dramatic decrease in rate of intramolecular transfer by three orders of magnitude compared to HSOWT, the rate of electron transfer was found to be 3 times faster in SDHR55Q relative to the wild type enzyme. These results indicate that Arg-55 is not involved in the pathway of electron transfer between the Mo and heme centres, but rather assists with the the docking of the heme group in HSO. As this process is not required in SDH, our results suggest that intramolecular electron transfer (IET) in HSOR160Q decreases because it is crucial for docking of the heme domain. Through potentiometric redox titrations, the effect of the active site amino acid substitutions on both the Mo and Fe redox potentials was investigated. No significant change was determined for the MoVI/V redox potentials, however, the heme potentials for SDHWT and SDHR55K were 40 mV higher than those of the other variants, with the lowest potentials belonging to SDHR55M and SDHH57A. Of further interest was that the MoVI/V couple is significantly lower than the heme couple (175 mV vs 240 mV respectively) in SDHWT. It appears that the positive charge of the Arg is important in regulating the heme redox potentials and could thereby contribute to modulating enzymatic activity. When SDH was immobilised on a modified pyrolytic graphite electrode, stable and high catalytic currents were observed, indicating facile heterogeneous electron transfer between the enzyme and the electrode. This good electron transfer allowed the catalytic properties of SDH and its substituted enzymes to be investigated as a function of potential. A pH dependence ( 59 mV/pH) in the catalytic operating potential was noted for SDHWT and SDHR55K, which appears to follow the pH dependence of the MoVI/V couple. This catalytic potential is pH-independent in the R55M and H57A variants, where the catalytic operating potentials appeared to follow the FeIII/II redox couple. It is proposed that two distinct pathways of electron transfer from the Mo centre to the electrode are likely to exist. The first is direct transfer from the Mo centre to the electrode at lower potential (~ 175 mV) while the second proceeds via the heme group (320 mV). The pathway followed is determined by the oxidation state of the heme group. A slight difference in the electron transfer rates of these two processes was seen, with direct transfer (from Mo) being the faster, which accounts for the unusual peak shape noted in the voltammogram for SDHWT at high sulfite concentrations, where the rate of catalytic activity slows at a higher potentials despite the greater thermodynamic driving force. This work provides new insights into the mechanism of enzymatic sulfite oxidation. Arg-55 has been shown to play an important role in the catalytic functioning of SDH in both substrate affinity and product release. Unlike what has been previously proposed, Arg-55 does not play a part in the pathway of electron transfer, but is rather involved in the regulation of the redox potentials of the metal centres in the enzyme.

This thesis is focussed on structural and functional studies of a novel glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. GpdQ is highly promiscuous and is the first known phosphatase which is capable of degrading all three classes of phosphate esters (mono-, di- and triesters). Remarkably, GpdQ is also able to hydrolyse stable aliphatic phosphate esters and has been shown to degrade the hydrolysis product of the nerve agent VX. For these reasons, GpdQ has been realised to have potential as a powerful bioremediator for the removal of organophosphate pesticides and nerve agents. GpdQ is a binuclear metallohydrolase in which one of the metal ions is very weakly bound. Chapter 1 introduces the catalytic mechanisms of binuclear metallohydrolases by examining two related phosphate ester-degrading enzymes. Since one of the main features of catalysis addressed in this thesis are the differential metal binding affinities of GpdQ, Chapter 1 also canvasses a range of other binuclear metallohydrolases with similar behaviour. Chapter 2 examines the structural and evolutionary relationship between GpdQ and a number of other related enzymes. Using genome database searches, the two most closely related enzymes are identified. In performing these searches, a novel, putative binuclear metallohydrolase from Homo sapiens is also discovered. This enzyme, Hsa_aTRACP, is most closely related to PAPs, however construction of a homology model indicates that the active site tyrosine residue of PAP is replaced by histidine. In this respect, it may represent an evolutionary link to Ser/Thr protein phosphatases and GpdQ. The biology and chemistry of this putative enzyme is discussed. PAPs are the only binuclear enzymes with an established heterovalent active site of the type Fe(III)-M(II) (where M=Fe, Zn or Mn) whereas the majority of enzymes in this family have homovalent metal centres, including GpdQ and Ser/Thr protein. This is brought about due to the nature of the coordination sphere imposed by the enzyme. The activity of GpdQ can be reconstituted in the presence of Co(II), Zn(II), Mn(II) and Cd(II). Chapter 3 examines the kinetic properties of a binuclear homovalent system by studying the kinetic properties of Cd(II)-substituted GpdQ and a corresponding model complex. This comparative study leads to the identification of a terminal hydroxide molecule as the likely reaction-initiating nucleophile in Cd(II)-GpdQ with a pKa of 9.4. In Chapter 4, a detailed study of the structural, kinetic and spectroscopic behaviour of Co(II)-substituted GpdQ is presented. This chapter specifically probes the formation of the binuclear active site, the role of the metal ions in catalysis, the identity of the nucleophile and the potential role of any first or second coordination sphere residues in the regulation of enzyme activity, proton donation and metal ion coordination. Based on these findings, a detailed reaction mechanism is proposed in which the substrate itself promotes the formation of the catalytically competent binuclear centre and phosphorolysis occurs following nucleophilic attack by a terminal hydroxide molecule. A potential role of Asn80 (a ligand of one of the metal ions) in regulating both substrate and metal binding, and the role of the bridging hydroxide molecule in the activation of the terminal nucleophile is proposed. Chapter 5 employs a combination of kinetic and spectroscopic techniques to probe the proposed catalytic mechanism of GpdQ in depth. The formation of the catalytically competent binuclear centre is observed in pre-steady state studies, an integral first step in the catalytic mechanism. The dissociation and rate constants associated with formation of the binuclear centre are quantified. The rate of substrate turnover in GpdQ is relatively modest but is enhanced by a structural rearrangement involving the flexible Asn80 ligand. This structural change fine-tunes the reaction mechanism, leading to optimal reactivity. The steady-state kinetic properties of a series of metal ion derivatives (Co(II), Cd(II) and Mn(II)) of GpdQ and their reactivity towards a number of substrates are also compared. These findings lead to the conclusion that the reaction mechanism of GpdQ is modulated by both substrate and metal ion. In this respect, GpdQ is adaptive to the environmental conditions to which it is exposed by employing a flexible mechanistic strategy to achieve catalysis. Chapter 6 correlates the electronic and geometric structure of the binuclear centre in GpdQ as a means to probe specific aspects of the mechanism. This study uses the wild type enzyme and a site-directed mutant (Asn80Asp) to examine the structure of the metal ions at two stages of catalysis. The role of the bridging hydroxide molecule in nucleophilic activation is specifically addressed by monitoring changes in the electronic exchange interaction and other structural parameters as a result of phosphate binding. Also, the coordination environment of the metal ions in both the free enzyme and the phosphate-bound enzyme of wild type and Asn80Asp GpdQ were assessed against the currently proposed structures. The findings in this chapter corroborate the proposed catalytic mechanism of GpdQ. In summary, this project led to a detailed understanding of the mechanism of GpdQ, and provided insight into how both the metal ion composition and the identity of the substrate may modulate this mechanism. The knowledge gained may lead to the design of catalytically more efficient derivatives (mutants) of GpdQ for application in bioremediation.

This thesis is focussed on structural and functional studies of a novel glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. GpdQ is highly promiscuous and is the first known phosphatase which is capable of degrading all three classes of phosphate esters (mono-, di- and triesters). Remarkably, GpdQ is also able to hydrolyse stable aliphatic phosphate esters and has been shown to degrade the hydrolysis product of the nerve agent VX. For these reasons, GpdQ has been realised to have potential as a powerful bioremediator for the removal of organophosphate pesticides and nerve agents. GpdQ is a binuclear metallohydrolase in which one of the metal ions is very weakly bound. Chapter 1 introduces the catalytic mechanisms of binuclear metallohydrolases by examining two related phosphate ester-degrading enzymes. Since one of the main features of catalysis addressed in this thesis are the differential metal binding affinities of GpdQ, Chapter 1 also canvasses a range of other binuclear metallohydrolases with similar behaviour. Chapter 2 examines the structural and evolutionary relationship between GpdQ and a number of other related enzymes. Using genome database searches, the two most closely related enzymes are identified. In performing these searches, a novel, putative binuclear metallohydrolase from Homo sapiens is also discovered. This enzyme, Hsa_aTRACP, is most closely related to PAPs, however construction of a homology model indicates that the active site tyrosine residue of PAP is replaced by histidine. In this respect, it may represent an evolutionary link to Ser/Thr protein phosphatases and GpdQ. The biology and chemistry of this putative enzyme is discussed. PAPs are the only binuclear enzymes with an established heterovalent active site of the type Fe(III)-M(II) (where M=Fe, Zn or Mn) whereas the majority of enzymes in this family have homovalent metal centres, including GpdQ and Ser/Thr protein. This is brought about due to the nature of the coordination sphere imposed by the enzyme. The activity of GpdQ can be reconstituted in the presence of Co(II), Zn(II), Mn(II) and Cd(II). Chapter 3 examines the kinetic properties of a binuclear homovalent system by studying the kinetic properties of Cd(II)-substituted GpdQ and a corresponding model complex. This comparative study leads to the identification of a terminal hydroxide molecule as the likely reaction-initiating nucleophile in Cd(II)-GpdQ with a pKa of 9.4. In Chapter 4, a detailed study of the structural, kinetic and spectroscopic behaviour of Co(II)-substituted GpdQ is presented. This chapter specifically probes the formation of the binuclear active site, the role of the metal ions in catalysis, the identity of the nucleophile and the potential role of any first or second coordination sphere residues in the regulation of enzyme activity, proton donation and metal ion coordination. Based on these findings, a detailed reaction mechanism is proposed in which the substrate itself promotes the formation of the catalytically competent binuclear centre and phosphorolysis occurs following nucleophilic attack by a terminal hydroxide molecule. A potential role of Asn80 (a ligand of one of the metal ions) in regulating both substrate and metal binding, and the role of the bridging hydroxide molecule in the activation of the terminal nucleophile is proposed. Chapter 5 employs a combination of kinetic and spectroscopic techniques to probe the proposed catalytic mechanism of GpdQ in depth. The formation of the catalytically competent binuclear centre is observed in pre-steady state studies, an integral first step in the catalytic mechanism. The dissociation and rate constants associated with formation of the binuclear centre are quantified. The rate of substrate turnover in GpdQ is relatively modest but is enhanced by a structural rearrangement involving the flexible Asn80 ligand. This structural change fine-tunes the reaction mechanism, leading to optimal reactivity. The steady-state kinetic properties of a series of metal ion derivatives (Co(II), Cd(II) and Mn(II)) of GpdQ and their reactivity towards a number of substrates are also compared. These findings lead to the conclusion that the reaction mechanism of GpdQ is modulated by both substrate and metal ion. In this respect, GpdQ is adaptive to the environmental conditions to which it is exposed by employing a flexible mechanistic strategy to achieve catalysis. Chapter 6 correlates the electronic and geometric structure of the binuclear centre in GpdQ as a means to probe specific aspects of the mechanism. This study uses the wild type enzyme and a site-directed mutant (Asn80Asp) to examine the structure of the metal ions at two stages of catalysis. The role of the bridging hydroxide molecule in nucleophilic activation is specifically addressed by monitoring changes in the electronic exchange interaction and other structural parameters as a result of phosphate binding. Also, the coordination environment of the metal ions in both the free enzyme and the phosphate-bound enzyme of wild type and Asn80Asp GpdQ were assessed against the currently proposed structures. The findings in this chapter corroborate the proposed catalytic mechanism of GpdQ. In summary, this project led to a detailed understanding of the mechanism of GpdQ, and provided insight into how both the metal ion composition and the identity of the substrate may modulate this mechanism. The knowledge gained may lead to the design of catalytically more efficient derivatives (mutants) of GpdQ for application in bioremediation.

Thesis (Ph.D.)--McMaster University (Canada), 1992. / Source: Dissertation Abstracts International, Volume: 54-02, Section: B, page: 0826.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 128-141).

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 118-124).