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dinucleotide (FAD) cofactor of pig kidney medium-chain s pecific acyl-coenzyme A (CoA) dehydrogenase (MCADH) has been replaced by ri bityl-3'-deoxy-FAD and ribityl-2'-deoxy-FAD. 3'-Deoxy-FAD-MCADH has propert ies very similar to those of native MCADH, indicating that the FAD-ribityl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 bindin g or catalysis. 2'-Deoxy-FAD-MCADH was characterized using the natural subs trate 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 su bstantial 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 corre sponds 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) Biochemis try 37, 1848-1860]. The difference of this effect of approximate to 6 pK un its (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 ca used by the interaction with the FAD-2'-OH. This energetic parameter derive d from the kinetic experiments (stabilization of transition state) is in ag reement with those obtained from static experiments (lowering of alpha C-H microscopic pK(a) of analogue, i.e., stabilization of anionic transition-st ate analogue). The contributions of the mio single H-bonds involved in subs trate 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 un ambiguously that the FAD cofactor and the substrate/product bind in an iden tical fashion, implying that the observed effects are mainly due to (the ab sence of) the FAD-ribityl-2'-OH hydrogen bond. The large energy associated with the 2'-OH hydrogen bond interaction is interpreted as resulting from t he changes in charge and the increased hydrophobicity induced by binding of lipophilic substrate. This is the first example demonstrating the direct i nvolvement of a flavin cofactor side chain in catalysis.