| Research Abstract |
The results of this research project were summarized as follows.
(1) NAADPH-adrenodoxin reductase
^<13>C-NMR spectra of this enzyme were observed by using ^<13>C-labeled FAD. In the complex of the enzyme with NADP, the signals for 4- and 10a-^<13>C shifted to up-fields by 2.1 and 0.7 ppm, respectively, and the 4a-^<13>C signal shifted to a down-field by 1.4 ppm in comparison with those of the free enzyme. These results suggest that the in the complex the pyridine moietly of NADP is located in the vicinity of C(4a)-C(4) region and that the pi-electron density of the 4a-position of FAD is decreased in the enzyme-NADP complex. This argues in favor of the electron transfer from NADPH to FAD of the enzyme.
(2) old yellow enzyme
The enzyme was reconstituted with an active site probe, 8-fluoro-8-demethyl FMN. When the reconstituted enzyme was subjected to specific limited proteolysis with bovine alpha-chymotrypsin, it was separated to 14K and 34k fragments and then the coenzyme, FMN, was covalent
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ly bound to the N-terminal leucine of the 14K fragment. These results suggest that the 14K domain is the flavin-binding domain and the coenzyme is located near the N-terminal region of the 14K domain.
(3) medium-chain acyl-CoA dehydrogenase
This enzyme was studied by ^<13>C-and ^<15>N- NMR, and resonance Raman spectroscopies. In the ^<13>C- and ^<15>N-NMR spectra of the complex of the enzyme with acetoacetyl-CoA, 5-^<15>N resonance of FAD was specifically upfield-shifted from that of the free enzyme, while other resonances were not appreciably shifted. This suggests that acetoacetyl-CoA induces specific change in the electronic state at N(5) of FAD. Resonance Raman spectra of the complex of the enzyme with acetoacetyl-CoA and of the purple complex formed upon the addition of octanoyl-CoA to the enzyme showed that in the ligands of the complexes a large contribution of ionie resonance structures was C(2)=C(1)-O. This fact probably facilitates the abstraction of the alpha-proton of substrates. Less
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