Role of Aromatic Stacking Interactions in the Modulation of the Two-Electron Reduction Potentials of Flavin and Substrate/Product in Megasphaera elsdenii Short-Chain Acyl-Coenzyme A Dehydrogena
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- 作者：Jackson D. Pellett ; Donald F. Becker ; Amy K. Saenger ; James A. Fuchs ; and Marian T. Stankovich
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：June 26, 2001
- 年：2001
- 卷：40
- 期：25
- 页码：7720 - 7728
- 全文大小：103K
- 年卷期：v.40,no.25(June 26, 2001)
- ISSN：1520-4995
文摘
The effects of aromatic stacking interactions on the stabilization of reduced flavin adeninedinucleotide (FAD) and substrate/product have been investigated in short-chain acyl-coenzyme Adehydrogenase (SCAD) from Megasphaera elsdenii. Mutations were made at the aromatic residues Phe160and Tyr366, which flank either face of the noncovalently bound flavin cofactor. The electrochemicalproperties of the mutants were then measured in the presence and absence of a butyryl-CoA/crotonyl-CoA mixture. Results from these redox studies suggest that the phenylalanine and tyrosine both engagein favorable 
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interactions with the isoalloxazine ring of the flavin to help stabilize formation of theanionic flavin hydroquinone. Disruption of these interactions by replacing either residue with a leucine(F160L and Y366L) causes the midpoint potential for the oxidized/hydroquinone couple (Eox/hq) to shiftnegative by 44-54 mV. The Eox/hq value was also found to decrease when aromatic residues containingelectron-donating heteroatoms were introduced at the 160 position. Potential shifts of -32 and -43 mVfor the F160Y and F160W mutants, respectively, are attributed to increased - repulsive interactionsbetween the ring systems. This study also provides evidence for thermodynamic regulation of the substrate/product couple in the active site of SCAD. Binding to the wild-type enzyme caused the midpoint potentialfor the butyryl-CoA/crotonyl-CoA couple (EBCoA/CCoA) to shift 14 mV negative, stabilizing the oxidizedproduct. Formation of product was found to be even more favorable in complexes with the F160Y andF160W mutants, suggesting that the electrostatic environment around the flavin plays a role in substrate/product activation.
- interactions with the isoalloxazine ring of the flavin to help stabilize formation of theanionic flavin hydroquinone. Disruption of these interactions by replacing either residue with a leucine(F160L and Y366L) causes the midpoint potential for the oxidized/hydroquinone couple (Eox/hq) to shiftnegative by 44-54 mV. The Eox/hq value was also found to decrease when aromatic residues containingelectron-donating heteroatoms were introduced at the 160 position. Potential shifts of -32 and -43 mVfor the F160Y and F160W mutants, respectively, are attributed to increased - repulsive interactionsbetween the ring systems. This study also provides evidence for thermodynamic regulation of the substrate/product couple in the active site of SCAD. Binding to the wild-type enzyme caused the midpoint potentialfor the butyryl-CoA/crotonyl-CoA couple (EBCoA/CCoA) to shift 14 mV negative, stabilizing the oxidizedproduct. Formation of product was found to be even more favorable in complexes with the F160Y andF160W mutants, suggesting that the electrostatic environment around the flavin plays a role in substrate/product activation.