Mechanism of activation of acyl-CoA substrates by medium chain acyl-CoA dehydrogenase: Interaction of the thioester carbonyl with the flavin adenine dinucleotide ribityl side chain

The flavin adenine dinucleotide (FAD) cofactor of pig kidney medium-chain specific acyl-coenzyme A (CoA) dehydrogenase (MCADH) has been replaced by ribityl-3'-deoxy-FAD and ribityl-2'-deoxy-FAD. 3'-Deoxy-FAD-MCADH has properties very similar to those of native MCADH, indicating that the FAD-ribityl side-chain 3'-OH group does not play any particular role in cofactor binding or catalysis. 2'-Deoxy-FAD-MCADH was characterized using the natural substrate C(8)CoA as well as various substrate and transition-state analogues. Substrate dehydrogenation in 2'-deoxy-FAD-MCADH is approximate to 1.5 x 10(7)-fold slower than that of native MCADH, indicating that disruption of the hydrogen bond between 2'-OH and substrate thioester carbonyl leads to a substantial transition-state destabilization equivalent to approximate to 38 kJ mol(-1). The alpha C-H microscopic pK(a) of the substrate analogue 3S-C(8)CoA, which undergoes alpha-deprotonation on binding to MCADH, is lowered from approximate to 16 in the free state to approximate to 11 (+/-0.5) when bound to 2'-deoxy-FAD-MCADH. This compares with a decrease of the same pK(a) to approximate to 5 in the complex with unmodified hwtMCADH, which corresponds to a pK shift of approximate to 11 pK units, i.e., approximate to 65 kJ mol(-1) [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The difference of this effect of approximate to 6 pK units (approximate to 35 kJ mol(-1)) between MCADH and 2'-deoxy-FAD-MCADH is taken as the level of stabilization of the substrate carbanionic species caused by the interaction with the FAD-2'-OH. This energetic parameter derived from the kinetic experiments (stabilization of transition state) is in agreement with those obtained from static experiments (lowering of alpha C-H microscopic pK(a) of analogue, i.e., stabilization of anionic transition-state analogue). The contributions of the mio single H-bonds involved in substrate activation (Glu376amide-N-H and ribityl-2'-OH) thus appear to behave additively toward the total effect. The crystal structures of native pMCADH and of 2'-deoxy-FAD-MCADH complexed with octanoyl-CoA/octenoyl-CoA show unambiguously that the FAD cofactor and the substrate/product bind in an identical fashion, implying that the observed effects are mainly due to (the absence of) the FAD-ribityl-2'-OH hydrogen bond. The large energy associated with the 2'-OH hydrogen bond interaction is interpreted as resulting from the changes in charge and the increased hydrophobicity induced by binding of lipophilic substrate. This is the first example demonstrating the direct involvement of a flavin cofactor side chain in catalysis.