Graduate Thesis Or Dissertation

Active site studies and design of ligands for affinity column separation of 2,5-dihydroxyacetanilide epoxidase (DHAE) I and II

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  • 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.
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