| Summary: | 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
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