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

Abstract

The flavin adenine dinucleotide (FAD) cofactor of pig kidney medium-chain specific acylcoenzyme A (CoA) dehydrogenase (MCADH) has been replaced by ribityl-3′-deoxy-FAD and ribityl-2′-deoxy-FAD. 3′-Deoxy-FAD-MCADH has properties very similar to those of native MCADH, indicating that the FAD-ribityl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 ≈1.5 × 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 ≈38 kJ mol -1 . The RC-H microscopic pK a of the substrate analogue 3S-C 8 CoA, which undergoes R-deprotonation on binding to MCADH, is lowered from ≈16 in the free state to ≈11 ((0.5) when bound to 2′-deoxy-FAD-MCADH. This compares with a decrease of the same pK a to ≈5 in the complex with unmodified hwtMCADH, which corresponds to a pK shift of ≈11 pK units, i.e., ≈65 kJ mol -1 [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The difference of this effect of ≈6 pK units (≈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 RC-H microscopic pK a of analogue, i.e., stabilization of anionic transition-state analogue). The contributions of the two 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.

Structures of Isobutyryl-CoA Dehydrogenase and Enzyme-Product Complex

“…The corresponding distances in MCAD are also 2.8 and 2.9 Å, respectively (14). These interactions are not only responsible for proper orientation of the substrate, they are also crucial for polarization of the substrate and the lowering of the pK a of the C-2 proton for abstraction by the catalytic base, Glu-376 (32). A water molecule is conserved in the binding cavity of all IBD subunits and is also present in the SCAD binding cavity.…”

Altered Balance of Half-reactions in p-Hydroxybenzoate Hydroxylase Caused by Substituting the 2′-Carbon of FAD with Fluorine

“…However, several enzymes have been identified (3,(5)(6)(7)(8) in which the 2Ј-hydroxyl of the ribityl chain appears to play an important role augmenting the redox chemistry. PHBH clearly belongs to this group of enzymes.…”

“…The redox properties of the free artificial flavin are virtually identical to those of natural flavin (3), suggesting that the introduction of a probe nucleus at the ribityl 2Ј-position has no significant effect on flavin solution chemistry. Surprisingly, although the fluorine probe is chemically isolated from the isoalloxazine reaction center, the catalytic and redox properties of several flavoenzymes are significantly different when the natural flavin is replaced with the 2Ј-F arabino analog or other 2Ј-derivatives (3)(4)(5)(6)(7)(8). These important changes indicate that the 2Ј-hydroxyl of natural flavins plays an important chemical role as a hydrogen-bonding active site substituent much like a serine residue might.…”

A Novel 3-Sulfinopropionyl Coenzyme A (3SP-CoA) Desulfinase from Advenella mimigardefordensis Strain DPN7 T Acting as a Key Enzyme during Catabolism of 3,3′-Dithiodipropionic Acid Is a Member of the Acyl-CoA Dehydrogenase Superfamily

“…This mode of substrate binding seems to be a general feature of all described crystal structures of Acds (1). Engst et al showed previously that the 2=-OH group not only promotes the initial ␣C-H deprotonation in MCAD but also supports proper substrate orientation (69). Hence, we propose that the cofactor is stabilizing the quaternary structure of Acd DPN7 and is necessary for correct positioning of 3SP-CoA.…”