Protonic Equilibria in the Reductive Half-Reaction of the Medium-Chain Acyl-CoA Dehydrogenase

Rudik, Irina; Ghisla, Sandro; Thorpe, Colin

doi:10.1021/bi980388z

Cited by 31 publications

(64 citation statements)

References 49 publications

(147 reference statements)

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“…However, rapid reaction experiments showed that the rate of deprotonation of (4-nitrophenyl)-acetyl-CoA by medium-chain acyl-CoA dehydrogenase decreases at higher pH (pK a ) 7.8) (49). Also, the polarization of cinnamoyl-CoA due to ionization of Glu376 decreases at higher pH (pK a ) 8.5) (50). In the case of GCD, polarization may be required not only to decrease the pK a of the R-proton of the substrate (41) but also to stabilize the intermediate crotonyl-CoA anion by delocalizing negative charge.…”

Section: Discussionmentioning

confidence: 99%

Proton Abstraction Reaction, Steady-State Kinetics, and Oxidation−Reduction Potential of Human Glutaryl-CoA Dehydrogenase

et al. 2000

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Glutaryl-CoA dehydrogenase catalyzes the oxidation of glutaryl-CoA to crotonyl-CoA and CO(2) in the mitochondrial degradation of lysine, hydroxylysine, and tryptophan. We have characterized the human enzyme that was expressed in Escherichia coli. Anaerobic reduction of the enzyme with sodium dithionite or substrate yields no detectable semiquinone; however, like other acyl-CoA dehydrogenases, the human enzyme stabilizes an anionic semiquinone upon reduction of the complex between the enzyme and 2,3-enoyl-CoA product. The flavin potential of the free enzyme determined by the xanthine-xanthine oxidase method is -0.132 V at pH 7.0, slightly more negative than that of related flavoprotein dehydrogenases. A single equivalent of substrate reduces 26% of the dehydrogenase flavin, suggesting that the redox equilibrium on the enzyme between substrate and product and oxidized and reduced flavin is not as favorable as that observed with other acyl-CoA dehydrogenases. This equilibrium is, however, similar to that observed in isovaleryl-CoA dehydrogenase. Comparison of steady-state kinetic constants of glutaryl-CoA dehydrogenase with glutaryl-CoA and the alternative substrates, pentanoyl-CoA and hexanoyl-CoA, suggests that the gamma-carboxyl group of glutaryl-CoA stabilizes the enzyme-substrate complex by at least 5.7 kJ/mol, perhaps by interaction with Arg94 or Ser98. Glu370 is positioned to function as the catalytic base, and previous studies indicate that the conjugate acid of Glu370 also protonates the transient crotonyl-CoA anion following decarboxylation [Gomes, B., Fendrich, G. , and Abeles, R. H. (1981) Biochemistry 20, 3154-3160]. Glu370Asp and Glu370Gln mutants of glutaryl-CoA dehydrogenase exhibit 7% and 0. 04% residual activity, respectively, with human electron-transfer flavoprotein; these mutations do not grossly affect the flavin redox potentials of the mutant enzymes. The reduced catalytic activities of these mutants can be attributed to reduced extent and rate of substrate deprotonation based on experiments with the nonoxidizable substrate analogue, 3-thiaglutaryl-CoA, and kinetic experiments. Determination of these fundamental properties of the human enzyme will serve as the basis for future studies of the decarboxylation reaction which is unique among the acyl-CoA dehydrogenases.

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Section: Discussionmentioning

confidence: 99%

Proton Abstraction Reaction, Steady-State Kinetics, and Oxidation−Reduction Potential of Human Glutaryl-CoA Dehydrogenase

et al. 2000

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“…CoASH (sodium salt) was purchased from Pharma-Waldhof (D-40549 Düsseldorf-Oberkassel, Germany), 4-nitrophenylacetone from Lancaster, and 4-aminophenylacetic acid from Fluka. Substrates C 4 -to C 16 CoA were either purchased from Sigma or prepared according to refs 29, 30 and purified by preparative HPLC. 4-Nitrophenylacetyl-CoA was obtained as described elsewhere (14).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 1998

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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.

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“…Isomerization is accompanied by a marked polarization of these chromophores (see later). Further, studies with 4‐thia‐enoyl‐CoA analogs showed that polarization is accompanied by proton uptake as the p K of the catalytic base is elevated markedly from ≈ 6.0 (in free MCAD [35]) to ≈ 9.2 in the product complex [31]. The next step (K 3 ) encompasses the chemistry of α,β‐dehydrogenation; k 3 corresponds to the transfer of two redox equivalents to the flavin, i.e., to its reduction, and is rate limiting with many poor substrates.…”

Section: Kinetic Mechanism Of the αβ‐Dehydrogenationmentioning

confidence: 99%

Acyl‐CoA dehydrogenases

Ghisla

Thorpe

2004

European Journal of Biochemistry

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307

297

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Acyl-CoA dehydrogenases constitute a family of flavoproteins that catalyze the a,b-dehydrogenation of fatty acid acyl-CoA conjugates. While they differ widely in their specificity, they share the same basic chemical mechanism of a,b-dehydrogenation. Medium chain acyl-CoA dehydrogenase is probably the best-studied member of the class and serves as a model for the study of catalytic mechanisms. Based on medium chain acyl-CoA dehydrogenase we discuss the main factors that bring about catalysis, promote specificity and determine the selective transfer of electrons to electron transferring flavoprotein. The mechanism of a,bdehydrogenation is viewed as a process in which the substrate aC-H and bC-H bonds are ruptured concertedly, the first hydrogen being removed by the active center base Glu376-COO -as an H + , the second being transferred as a hydride to the flavin N(5) position. Hereby the pK a of the substrate aC-H is lowered from > 20 to % 8 by the effect of specific hydrogen bonds. Concomitantly, the pK a of Glu376-COO -is also raised to 8-9 due to the decrease in polarity brought about by substrate binding. The kinetic sequence of medium chain acyl-CoA dehydrogenase is rather complex and involves several intermediates. A prominent one is the molecular complex of reduced enzyme with the enoyl-CoA product that is characterized by an intense charge transfer absorption and serves as the point of transfer of electrons to the electron transferring flavoprotein. These views are also discussed in the context of the accompanying paper on the three-dimensional properties of acyl-CoA dehydrogenases.

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Protonic Equilibria in the Reductive Half-Reaction of the Medium-Chain Acyl-CoA Dehydrogenase

Cited by 31 publications

References 49 publications

Proton Abstraction Reaction, Steady-State Kinetics, and Oxidation−Reduction Potential of Human Glutaryl-CoA Dehydrogenase

Proton Abstraction Reaction, Steady-State Kinetics, and Oxidation−Reduction Potential of Human Glutaryl-CoA Dehydrogenase

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

Acyl‐CoA dehydrogenases

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