Substrate Activation by Acyl-CoA Dehydrogenases: Transition-State Stabilization and pKs of Involved Functional Groups

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Abstract
The mechanism by which acyl-CoA dehydrogenases initiate catalysis was studied by using p-substituted phenylacetyl-CoAs (substituents -NO2, -CN, and CH3CO-), 3S-C8-, and 3‘-dephospho-3S-C8CoA. These analogues lack a βC−H and cannot undergo α,β-dehydrogenation. Instead they deprotonate at αC−H at pH ≥ 14 to form delocalized carbanions having strong absorbancies in the near UV−visible spectrum. The pKas of the corresponding phenylacetone analogues were determined as ≈13.6 (-NO2), ≈14.5 (-CN), and ≈14.6 (CH3CO-). Upon binding to human wild-type medium-chain acyl-CoA dehydrogenase (MCADH), all analogues undergo αC−H deprotonation. While the extent of deprotonation varies, the anionic products form charge-transfer complexes with the oxidized flavin. From the pH dependence of the dissociation constants (Kd) of p-NO2-phenylacetyl-CoA (4NPA-CoA), 3S-C8-CoA, and 3‘-dephospho-3S-C8CoA, four pKas at ≈5, ≈6, ≈7.3, and ≈8 were identified. They were assigned to the following ionizations: (a) pKa ≈5, ligand (L−H) in the MCADH∼ligand complex; (b) pKa ≈6, Glu376-COOH in uncomplexed MCADH; (c) pKa ≈7.3, Glu99-COOH in uncomplexed MCADH (Glu99 is a residue that flanks the bottom of the active-center cavity; this pK is absent in the mutant Glu99Gly-MCADH); and (d) pK ≈8, Glu99-COOH in the MCADH∼4NPA-CoA complex. The pKa ≈6 (b) is not significantly affected in the MCADH∼4NPA-CoA complex, but it is increased by ≥1 pK unit in that with 3S-C8CoA and further in the presence of C8-CoA, the best substrate. The αC−H pKas of 4NPA-CoA, of 3S-C8-CoA, and of 3‘-dephospho-3S-C8CoA in the complex with MCADH are ≈5, ≈5, and ≈6. Compared to those of the free species these pKa values are therefore lowered by 8 to ≥11 pH units (50 to ≥ 65 kJ mol-1) and are close to the pKa of Glu376-COOH in the complex with substrate/ligand. This effect is ascribed mainly to the hydrogen-bond interactions of the thioester carbonyl group with the ribityl-2‘-OH of FAD and Glu376-NH. It is concluded that the pKa shifts induced with normal substrates such as n-octanoyl-CoA are still higher and of the order of 9−13 pK units. With 4NPA-CoA and MCADH, αC−H abstraction is fast (kapp ≈55 s-1 at pH 7.5 and 25 °C, deuterium isotope effect ≈1.34). However, it does not proceed to completion since it constitutes an approach to equilibrium with a finite rate for reprotonation in the pH range 6−9.5. The extent of deprotonation and the respective rates are pH-dependent and reflect apparent pKas of ≈5 and ≈7.3, which correspond to those determined in static experiments.

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