Substrate activation by acyl-CoA dehydrogenases:: Transition-state stabilization and pKs of involved functional groups

The mechanism by which acyl-CoA dehydrogenases initiate catalysis was studied by using p-substituted phenylacetyl-CoAs (substituents -NO(2), -CN, and CH(3)CO-). 3S-C(8)-, and 3'-dephospho-3S-C(8)CoA. These analogues lack a beta C-H and cannot undergo alpha,beta-dehydrogenation. Instead they deprotonate at alpha C-H at pH greater than or equal to 14 to form delocalized carbanions having strong absorbancies in the near W-visible spectrum. The pK(a)s of the corresponding phenylacetone analogues were determined as approximate to 13.6 (-NO(2)), approximate to 14.5 (-CN), and approximate to 14.6 (CH(3)CO-). Upon binding to human wild-type medium-chain acyl-CoA dehydrogenase (MCADH), all analogues undergo alpha C-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 (K(d)) of p-NO(2)-phenylacetyl-CoA (4NPA-CoA), 3S-C(8)-CoA, and 3'-dephospho-3S-CsC(8)A, four pK(a)s at approximate to 5, approximate to 6, approximate to 7.3, and approximate to 8 were identified. They were assigned to the following ionizations: (a) pK(a) approximate to 5, ligand (L-H) in the MCADH similar to ligand complex (b) pK(a) approximate to 6, Glu376-COOH in uncomplexed MCADH; (c) pK(a) approximate to 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 approximate to 8, Glu99-COOH in the MCADH similar to 4NPA-CoA complex. The pK(a) approximate to 6 (b) is not significantly affected in the MCADH similar to 4NPA-CoA complex, but It is increased by greater than or equal to 1 pK unit in that with 3S-C(8)CoA and further in the presence of C(8)-CoA, the best substrate. The alpha C-H pK(a)s of 4NPA-CoA, of 3S-C(8)-CoA, and of 3'-dephospho-3S-C(8)CoA in the complex with MCADH are approximate to 5, approximate to 5, and approximate to 6. Compared to those of the free species these pK, values are therefore lowered by 8 to greater than or equal to 11 pH units (50 to greater than or equal to 65 kJ mol(-1)) and are close to the pK(a) 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. Tt is concluded that the pK(a) 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, alpha C-M abstraction is fast (k(app)approximate to 55 s(-1) at pH 7.5 and 25 degrees C, deuterium isotope effect approximate to 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 pK(a)s of approximate to 5 and approximate to 7.3, which correspond to those determined in static experiments.