A thesis submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy, Johannesburg, 1996 / The glutathione S-transferases (GST) are a supergene family of h0111o-or heterodimeric Phase II detoxification enzymes which catalyse the S-conjugation between glutathione and an electrophilic substrate. The active site can be divided into two adjacent functional regions; a highly specific Gssite for binding the physiological substrate glutathione and a nonspecific If-site for binding nonpolar electrophilic substrates. Unfolding of porcine class Pi isoenzyme (pGSTPl~l) was monitored under equilibrium conditions using different physicochemical parameters. The coincidence of unfolding curves obtained with functional and structural probes, the absence of thermodynamically stable intermediates such as a folded monomer, and the dependence of pGSTPl··l stability upon protein concentration, indicate a cooperative and concerted two-state unfolding transition between native dimeric pGSTPl-l and unfolded monomeric enzyme. Equilibrium and kinetic unfolding experiments employing tryptophan fluorescence and enzyme activity measurements were preformed to study the effect of ligand binding to the G-site on the unfolding and stability of the porcine class pi glutathione S-transferase against urea. The presence of glutathione caused a shift in the equilibrium-unfolding curves towards lower urea concentrations and enhanced the first-order rate constant for unfolding suggesting a destabilisation of the pGSTPl-l structure against urea. The presence of either glutathione sulphonate or S-hexylglutathione, however, produced the opposite effect in that their binding to the G-site appeared to exert a stabilising effect against urea. The binding of these glutathione analogues also reduced significantly the degree of cooperativity of unfolding indicating a possible change in the protein's unfolding pathway. / MT2017

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirement for the degree of Master of science Johannesburg. 1996 / An attempt has been made to purify and isolate a starch endo-hydrolase enzyme produced by Streptococcus sanguis, an organism that may be implicated in dental caries. In order to isolate the enzyme by affinity binding,& chemically modified amylopectin Was prepared, similar to the preparation of chromogenic substrate for the assay of a-arnylase, but without dye. The amylopectin was treated with 10 percent ammonium sulphate at 55°C for 45 minutes. A crude enzyme extract was prepared from concentrated culture medium and by precipitation with 60 percent ammonium sulphate. The concentrated culture medium was added to the modified amylopectin substrate and the mixture was incubate for 30 minutes at 37'C and centrifuged. The precipitate was resuspended in 20 mM bepes buffer pH 6.5 which contained 0.02 M KCI to release the enzyme from the enzyme-substrate complex. The suspension was tested for enzyme activity and the presence of proteins, More than 50 percent of the yield of the enzyme was achieved by this process, after three assays, from both crude enzyme extracts and enzyme serum samples. A 5.2 fold purification was obtained from the extraction process of the crude enzyme extract and a protective enzyme activity effect was noticed in the presence of ammonium sulphate. The analytical methods selected for the activity assay were mainly used for the activity evaluation of a-amylases and carbohydrate hydrolysing enzymes. The result showed carbohydrate interference. The isolation method proved sensitive and highly specific for the isolation of a starch metabolizing enzyme produced from Streptococcus sanguis 1MC 204. The purification of the enzyme by gel filtration and its characterization by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that this enzyme was a glycoprotein . SDS-PAGE was performed with mucin and heparin in the. presence of other. proteins as markers, and stained with the periodic acid- aldan prestained silver method. This gave one transparent-band around the phosphorylase b marker and four other more slowly running clear bands. Further, the comparison of scans of several proteins and glycoproteins with the scan of the eluted sample of the amylopectin extracted enzyme showed a similarity with the UV scan of mucin and confirmed that the enzyme was a glycoprotein. It may be further characterized by selecting methods that take into account the ambohydrate content of the protein and by eliminating the carbohydrate interference in the assays. 3

Coenzimas piridínicos desempenham papel fundamental relacionado à suas propriedades de óxido-redução. Assim, participam da cadeia respiratória, onde se situam, devido ao valor do potencial de óxido-redução do par, logo no início, sendo os primeiros intermediários entre certos substratos e oxigênio. Constituem-se também no primeiro local de síntese de ATP; muito importante, presumivelmente em conexão com a função respiratória, é de fato de os coenzimas piridínicos intervirem nos sistemas de algumas transhidrogenases. / Not available

Thesis (M.S.)--Chinese University of Hong Kong. / Includes bibliographical references.

Thesis (M. Phil.)--Chinese University of Hong Kong, 1976. / Bibliography: leaves 82-91.

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Cyclic peptides constitute an attractive class of compounds for drug development, however the numerous problems associated with their synthesis have limited their applicability. The cyclisation step itself is particularly problematic, with solution phase cyclisations being required to be conducted under very high dilution to promote cyclisation over unwanted side reactions such as oligomerisation. In addition, epimerisation, leading to the loss of chiral integrity at the terminal residues is a major concern. Attention is now turning to biochemical cyclisation strategies, such as SICLOPPS and sortase mediated ligation, although these also come with their own inherent disadvantages, for example, in the case of sortase mediated ligation, there is significant “scarring” of the target due to the presence of a four amino acid long recognition sequence. Cyclisation using ribosomally synthesised post translationally modified enzymes is also gaining popularity. One such family of enzymes is the patellamides. PatGmac is capable of performing cyclisations on linear peptide substrates with minimal scarring compared to the aforementioned alternatives and, importantly, with no epimerisation and could constitute a greener and more facile route to cyclic peptides. The work herein details some of the investigations designed to define the range of synthetic utility and test the flexibility of the enzymes. This was done qualitatively, by designing a variety of linear peptide analogues of the natural product, homophymine A, featuring unique structural moieties and evaluating their compatibility with the enzyme. It was also done quantitatively, using an LCMS based semi-quantitative strategy, to assess differences between similar, but different, enzymes and to assess whether there were differences in how different substrates are processed by the same enzyme. In addition to this, a variety of these substrates were also assessed for their proclivity to cyclise under standard chemical conditions for comparison. Lastly, with the increasing appearance cyclic peptides and non-peptidic macrocycles in the libraries of compounds being considered for clinical trials, there is now a growing need for computational modelling of these structures. Herein, a 3D v structure for the natural product callipetin N is proposed for the first time, determined using a combination of computational and NMR techniques.