Molecular Mechanism of the Drop in the pK_a of a Substrate Analog Bound to Medium-Chain Acyl-CoA Dehydrogenase: Implications for Substrate Activation

Abstract

The pK_a value of a substrate analogue 3-thiaoctanoyl-CoA at αC-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 pK_a value became [e. g., 7.4 (8-NH₂-FAD) and 4.0 (8-CN-FAD)], suggesting that the flavin ring itself affects the pK_a 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 pK_a by ca. 2.5 units. These results indicate that the interaction between the ligand and the flavin ring also serves to lower the pK_a of the ligand, in addition to the hydrogen bonds at C(1)=O of the ligand.