ABSTRACT: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 C-H and cannot undergo R, -dehydrogenation. Instead they deprotonate at RC-H at pH g 14 to form delocalized carbanions having strong absorbancies in the near UV-visible spectrum. The pK a s of the corresponding phenylacetone analogues were determined as ≈13.6 (-NO 2 ), ≈14.5 (-CN), and ≈14.6 (CH 3 CO-). Upon binding to human wild-type medium-chain acyl-CoA dehydrogenase (MCADH), all analogues undergo RC-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-C 8 CoA, four pK a s at ≈5, ≈6, ≈7.3, and ≈8 were identified. They were assigned to the following ionizations: (a) pK a ≈5, ligand (L-H) in the MCADH∼ligand complex; (b) pK a ≈6, Glu376-COOH in uncomplexed MCADH; (c) pK a ≈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 pK a ≈6 (b) is not significantly affected in the MCADH∼4NPA-CoA complex, but it is increased by g1 pK unit in that with 3S-C 8 CoA and further in the presence of C 8 -CoA, the best substrate. The RC-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 ≈5, ≈5, and ≈6. Compared to those of the free species these pK a values are therefore lowered by 8 to g11 pH units (50 to g 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. It 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, RC-H abstraction is fast (k app ≈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 pK a s of ≈5 and ≈7.3, which correspond to those determined in static experiments.Acyl-CoA dehydrogenases catalyze the R, -dehydrogenation of fatty acid acyl-CoA conjugates to their corresponding enoyl-CoA products; the redox equivalents formed in this reaction are transferred to electron transferring flavoprotein and further to the respiratory chain (1, 2). A peculiarity of the R, -dehydrogenation reaction is that it involves the concomitant fission of two kinetically stable C-H bonds. In the past, studies with medium-chain acyl-CoA dehydrogenase ...

The catalytically essential glutamate residue that initiates catalysis by abstracting the substrate R-hydrogen as H + is located at position 376 (mature MCADH numbering) on loop JK in medium chain acyl-CoA dehydrogenase (MCADH). In long chain acyl-CoA dehydrogenase (LCADH) and isovalerylCoA dehydrogenase (IVDH), the corresponding Glu carrying out the same function is placed at position 255 on the adjacent helix G. These glutamates thus act on substrate approaching from two opposite regions at the active center. We have implemented the topology of LCADH in MCADH by carrying out the two mutations Glu376Gly and Thr255Glu. The resulting chimeric enzyme, "medium-/long" chain acyl-CoA dehydrogenase (MLCADH) has ∼20% of the activity of MCADH and ∼25% that of LCADH with its best substrates octanoyl-CoA and dodecanoyl-CoA, respectively. MLCADH exhibits an enhanced rate of reoxidation with oxygen, however, with a much narrower substrate chain length specificity that peaks with dodecanoyl-CoA. This is the same maximum as that of LCADH and is thus significantly shifted from that of native MCADH (hexanoyl/octanoyl-CoA). The putative, common ancestor of LCADH and IVDH has two Glu residues, one each at positions 255 and 376. The corresponding MCADH mutant, Thr255Glu (glu/glu-MCADH), is as active as MCADH with octanoyl-CoA; its activity/chain length profile is, however, much narrower. The topology of the Glu as H + abstracting base seems an important factor in determining chain length specificity and reactivity in acyl-CoA dehydrogenases. The mechanisms underlying these effects are discussed in view of the three-dimensional structure of MLCADH, which is presented in the accompanying paper [Lee et al. (1996) Biochemistry 35, 12412-12420].Acyl-CoA dehydrogenases are a class of flavoproteins that catalyze the desaturation of acyl-CoA substrates. Four known members are involved in mammalian mitochondrial -oxidation of fatty acids (short-, medium-, long-, and very long chain acyl-CoA dehydrogenases), and three (isovaleryl-, isobutyryl-and glutaryl-CoA dehydrogenases) are involved in the degradation of amino acids. In addition microsomal or peroxisomal acyl-CoA oxidases and a variety of related enzymes of bacterial or plant origin can be viewed as sharing the capacity to catalyze the seemingly identical chemical process, the substrate dehydrogenation at the positions R, .

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.