Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Univ., Diss., 2009--Marburg. / Enth. Sonderdr. aus versch. Zeitschriften.

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A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg, 2014. / The seemingly simple proton abstraction reactions underpin many chemical transformations including isomerization reactions and are thus of immense biological significance. Despite the energetic cost, enzyme-catalyzed proton abstraction reactions show remarkable rate enhancements. The pathways leading to these accelerated rates are numerous and on occasion partly enigmatic. The isomerization of the steroid, Δ5- androstene-3,17-dione by the human glutathione transferase A3-3 in mammals was investigated to gain insight into the mechanism. Particular emphasis was placed on the nature of the transition state, the intermediate suspected of aiding this process and the hydrogen bonds postulated to be the stabilizing forces of these transient species. Kinetics studies on Δ5-androstene-17-one, a substrate that is incapable of forming hydrogen bonds reveal that such stabilizing forces are not a requirement to explain the observed rate enhancements. The UV-Vis detection of the intermediate places this specie in the catalytic pathway while fluorescence spectroscopy is used to obtain the binding constant of the intermediate analogue equilenin. Analysis of the kinetics data in terms of the Marcus formalism indicates that the human glutathione transferase A3-3 lowers the intrinsic kinetic barrier by 3 kcal/mole. The results lead to the conclusion that this reaction proceeds through an enforced concerted mechanism in which the barrier to product formation is kinetically insignificant.

A dissertation submitted in fulfilment of the requirements for the degree of Master of Science at the University of the Witwatersrand. Johannesburg February 1994 / Glutathione S-transferases are multifunctional intracellular proteins. They catalyse the conjugation of glutathione to endogenous'or foreign electrophiles, and also bind non-substrate ligands. Class Pi glutathione S-transferase (pGSTPl~l) was purified from porcine lung to a specific. activity of 6.63p.ffiol/min/mg. The homodimeric protein has a molecular weight of about 4~.7kD and an isoelectric point of 8.6. Anionic ligand-binding properties of this isoenzyme were investigated. Steady-state fluorescence methods were used to determine ~ values for 8-anilino··l~naphtha1enesulphonic acid (K, == 17.1p.M and 11.1J.tM using fluorescence enhancement techniques and quenching techniques respectively), bromosulphophtbalein (Kcl=1.1p.M at pH 6.5 and 2.4/jM at pH 7.5) and glutathione {~=1201I.M). The affinity of bromosulphophthalein for the enzyme, in the presence of 10mM glutathione was slightly enhanced (~=O.7.uM at pH 6.5). The energy transfer betwecz the protein's tryptophan residues and 8-anUino-l-naphthalene sulphonic acid was observed and found to be about 56% efficient. The impact of ligand binding on both protein structure and catalytic activity were assessed. Kinetic studies show that the active site of the enzyme is not the primary binding site for the non-substrate ligands, but that the binding of bromosulphophthalein and to a lesser extent 8~ani1ino-l-!.~phtha1ene sulphonic acid, does affect the active site of the enzyme, especially aner saturating concentrations of the ligand. This may be the result of a small ligand-induced conformational change. Fluorescence studies also indicate that the primary site for anionic ligand binding is not in close proximity to either Trp28 or Trp38 in domain I, Competition studies indicated that the two anionic ligands bind the Same site, < Prorein fluorescence, chemical modification « and size-exclusion HPLC data indicate that ligand binding does 110t induce gross conformational changes in the protein.

S-acylation, commonly known as S-palmitoylation, is a reversible posttranslational lipid modification in which fatty acid, usually palmitic acid, covalently attaches to specific cysteine residues of proteins via thioester bonds. Palmitoylation enhances the hydrophobicity of proteins and contributes to their membrane association. It plays roles in protein trafficking, signalling, protein-protein interaction, protein stability and other important cellular functions. A family of Protein S-acyl Transferases (PATs) is responsible for this reaction. PATs are multi-pass transmembrane proteins that possess a catalytic Asp- His-His-Cys cysteine rich domain (DHHC-CRD) of ~50 amino acids. In Arabidopsis there are at least 24 such DHHC-CRD containing PAT proteins and they are named as AtPAT01 to AtPAT24. The function of only 2 AtPATs, AtPAT10 and AtPAT24 were studied in some detail, and a recent survey showed the ubiquitous expression pattern and different membrane localization habit of all 24 AtPATs. However, the biological function of the remaining 22 AtPATs in Arabidopsis was not reported when I started my project. Therefore, we carried out an initial screen of all the available T-DNA insertion lines of the 22 Arabidopsis PATs and identified transcriptional null mutants of 18 of the AtPATs. Among them, the k/o mutant plants of only 3 genes showed significantly altered phenotypes compared to wild-type Arabidopsis, and the mutants are named as atpat14, atpat21 and plp1(PAT-like Protein 1). This project aims to characterize these three putative PATs in details in terms of their PAT activities, catalytic domains, expression patterns, subcellular localizations and biological functions. AtPAT14 was proved as a PAT by yeast complementary and in vitro auto-acylation assays. Mutagenesis studies clearly demonstrated that the cysteine residue in the DHHCmotif is essential for the enzyme activity of AtPAT14. Transgenic Arabidopsis plants expressing AtPAT14-GFP were observed and it was shown that AtPAT14 is predominantly localized at the Trans-Golgi. The phenotype was observed in both atpat14-1 and atpat14-2 mutant lines and this showed that the leaves of both lines were aging much faster than the WT. Analysis of the levels of different phytohormones revealed that the mutant leaves contained much higher salicylic acid (SA) than the WT. This coincided with the increased transcript levels of genes involved in SA biosynthesis and signalling. Therefore, AtPAT14 mediated protein S-acylation plays important roles in leaf senescence via the regulation of SA biosynthesis and signalling pathways. AtPAT21 was also confirmed as a PAT and the DHHC its functional domain by similar approaches as for AtPAT14. The plasma membrane (PM) localized AtPAT21 plays essential roles in both male and female gametogenesis. As such, loss-of-function by TDNA insertion in AtPAT21 leads to the plant being completely sterile. Therefore, AtPAT21-mediated S-acylation of proteins(s) plays important roles in the reproduction of Arabidopsis. AtPLP1 (PAT-like Protein 1) contains the signature DHHC-CRD. However, it does not rescue the growth defects of akr1, pfa3 and swf1, the 3 yeast PAT mutants used in enzyme activity assays of other known PATs from plant and animals. Further, the cysteine residue in the DHHC motif was not essential for the function of AtPLP1 as mutated variant containing serine in place of cysteine of the DHHC motif can still rescue the growth defects of atplp1-1. Seedling establishment of atplp1-1 was impaired without external carbon source. This is because the efficiency in converting the seed storage lipid to sugar in the mutant is much lower than WT due to the defective β-oxidation process involved in the degradation of free fatty acids released from lipid during post-germinative growth. In addition, atplp1-1 seedlings are also de-etiolated in the dark, and this was coincided with more cytokinin (CK) and less active gibberellin (GA) related pathway in the mutant. Other defects were also found in atplp1-1, such as hypersensitive to abscisic acid (ABA) and sugar during seed germination and abnormal shoot apical meristem (SAM) in older plants. Therefore, protein S-acyltransferases play distinct and diverse roles throughout the life cycle, from seed germination, seedling growth to seed production in Arabidopsis. This is most likely through the palmitoylation of an array of proteins they modify. Hence, our results provide vital clues for future studies on the molecular mechanism as to how AtPATs operate in plant.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.