J. Biochem, 2003, Vol. 134, No. 6 835-842
© 2003 Japanese Biochemical Society
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Molecular Mechanism of the Drop in the pKa of a Substrate Analog Bound to Medium-Chain Acyl-CoA Dehydrogenase: Implications for Substrate Activation
1 Department of Molecular Physiology and 2 Department of Molecular Enzymology, Graduate School of Medical Sciences, Kumamoto University, Honjo, Kumamoto 860-8556; and 3 Division of Biological Chemistry, Department of Life Science, Graduate School of Science and Technology, Kobe University, Nada-ku, Kobe 657-8501
The pKa value of a substrate analogue 3-thiaoctanoyl-CoA at 
C-H is known to drop from ca. 16 in the free state to 5–6 upon binding to medium-chain acyl-CoA dehydrogenase (MCAD). The molecular mechanism underlying this phenomenon was investigated by taking advantage of artificial FADs, i.e., 8-CN-, 7,8-Cl2-, 8-Cl-, 8-OCH3-, 8-NH2-, ribityl-2'-deoxy-8-CN-, and ribityl-2'-deoxy-8-Cl-FADs, reconstituted into MCAD. The stronger the electron-withdrawing ability of the substituent, the smaller the pKa value became [e.g., 7.4 (8-NH2-FAD) and 4.0 (8-CN-FAD)], suggesting that the flavin ring itself affects the pKa value of the ligand via a charge-transfer interaction with the ligand. The destruction of the hydrogen bond between the thioester C(1)=O and the ribityl-2'-OH of FAD raised the pKa by ca. 2.5 units. These results indicate that the interaction between the ligand and the flavin ring also serves to lower the pKa of the ligand, in addition to the hydrogen bonds at C(1)=O of the ligand.
* To whom correspondence should be addressed. Fax: +81-96-373-5052, E-mail: nishina{at}medic.kumamoto-u.ac.jp
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