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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.

A series of compounds, 7-8 and 20-25, were tested as competitive inhibitors of 2,5-dihydroxyacetanilide epoxidase I (DHAE I) and DHAE II. A Hammett plot was constructed for each enzyme to determine the effect of electron density on inhibition. DHAE I gave a linear, highly correlated plot (r²=0.91) that signifies the importance of the amide oxygen in 1 on substrate binding. The plot for DHAE 11 is curved showing the greatest degree of inhibition with 7 suggesting steric factors within the active site control substrate binding. From these data, we conclude that each enzyme binds substrate in an opposite fashion and that this alone controls the stereochemistry of epoxide formation in 2 and 3. Alternative substrates, 26-29 and 33, were also synthesized and tested for product formation. All compounds, except 29, were accepted as alternative substrates, although the rates varied significantly. Surprisingly, 33 was accepted as an alternative substrate of DHAE II suggesting that the conformation of the amide bond in 33 is similar to the conformation required for catalytic activity in this enzyme. This information was then used to design ligands for affinity column separation of DHAE I and DHAE II from their protein mixtures. 35 and 36 were synthesized and attached to carbonyl di-imidazole activated agarose. Column I was tested three times with DHAE I enzyme preparations. The first attempt did not result in active enzyme being eluted from the column. The second attempt maintained the resin in the oxidized state. Protein was found to elute very quickly: no protein was found after fraction 4. The third attempt resulted in active enzyme in fractions 4-23. Column 2 was used twice for the attempted isolation of DHAE II from its protein mixture. The second attempt for column 2 mirrored the results for the third attempt with column 1. Neither column resulted in homogeneous enzyme by SDS-PAGE. / Graduation date: 2003

Graduation date: 1994

A series of conformationally constrained and flexible pseudopeptides were prepared and their thermodynamic parameters on binding to the Grb2 SH2 domain were determined by isothermal titration calorimetry (ITC). Cyclopropane constrained analogs having hydrophobic amino acid residues at the pTyr+1 position exhibited, on average, a 2-5 fold improvement in binding affinities with the enhancement in affinities due to a more favorable enthalpy, not entropy, of binding. This serves as the first set of examples which demonstrate that favorable entropies of binding are not an inherent characteristic of ligand preorganization. Incorporation of polar amino acid residues at the pTyr+1 position eventuated in a slightly different thermodynamic effect than what was observed with the hydrophobic analogs. The constrained molecules exhibited greater binding affinities for the Grb2 SH2 domain and that increase in affinity was a consequence of a more favorable enthalpy, not entropy, of binding. However, the binding entropies for the polar set of constrained and flexible molecules were all negative. Structural information obtained from the co-crystallization of selected constrained and flexible ligand pairs with the Grb2 SH2 domain revealed an increase in conformational mobility of the BC loop in the complexes of the constrained derivatives and the presence of a greater number of direct polar contacts, but fewer water-mediated interactions between the phosphate group of the constrained molecules and the pTyr binding pocket of the domain. The construction of the cis-hydrindanone ring system in sieboldine A was accomplished utilizing either a Lewis acid-mediated silyl-directed Nazarov cyclization of a functionalized divinyl ketone or a sequential Ni(0)-catalyzed 1,4-addition/5-endo-dig cyclization. Propargylzinc bromide was shown to undergo conjugate addition to the [alpha]-aminopropyl substituted enone using Ni(acac)₂, thus providing a new, mild protocol for the conjugate propargylation reaction. Further efforts toward the formation of the α-epoxy ketone are described. / text