Role of the Carbonyl Group in Thioester Chain Length Recognition by the Medium Chain Acyl-CoA Dehydrogenase

Trievel, Raymond C.; Wang, Rong; Anderson, Vernon E.; Thorpe, Colin

doi:10.1021/bi00027a009

Cited by 35 publications

(84 citation statements)

References 46 publications

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“…Therefore, as demonstrated in the literature, VLCAD not only allows longer chain-length substrates to bind, but it prefers them (4). It has been shown for MCAD that substrate/ product binding energy increases linearly with chain length (390 cal/CH 2 group) (42). For ACADs, product release is the rate-limiting step (43).…”

Section: Resultsmentioning

confidence: 99%

Structural Basis for Substrate Fatty Acyl Chain Specificity

McAndrew

Wang

Mohsen

et al. 2008

Journal of Biological Chemistry

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Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a member of the family of acyl-CoA dehydrogenases (ACADs). Unlike the other ACADs, which are soluble homotetramers, VLCAD is a homodimer associated with the mitochondrial membrane. VLCAD also possesses an additional 180 residues in the C terminus that are not present in the other ACADs. We have determined the crystal structure of VLCAD complexed with myristoyl-CoA, obtained by co-crystallization, to 1.91-Å resolution. The overall fold of the N-terminal ϳ400 residues of VLCAD is similar to that of the soluble ACADs including medium-chain acyl-CoA dehydrogenase (MCAD). The novel C-terminal domain forms an ␣-helical bundle that is positioned perpendicular to the two N-terminal helical domains. The fatty acyl moiety of the bound substrate/product is deeply imbedded inside the protein; however, the adenosine pyrophosphate portion of the C14-CoA ligand is disordered because of partial hydrolysis of the thioester bond and high mobility of the CoA moiety. The location of Glu-422 with respect to the C2-C3 of the bound ligand and FAD confirms Glu-422 to be the catalytic base. In MCAD, Gln-95 and Glu-99 form the base of the substrate binding cavity. In VLCAD, these residues are glycines (Gly-175 and Gly-178), allowing the binding channel to extend for an additional 12 Å and permitting substrate acyl chain lengths as long as 24 carbons to bind. VLCAD deficiency is among the more common defects of mitochondrial ␤-oxidation and, if left undiagnosed, can be fatal. This structure allows us to gain insight into how a variant VLCAD genotype results in a clinical phenotype. Very-long-chain acyl-CoA dehydrogenase (VLCAD)3 is one of five acyl-CoA dehydrogenases (ACADs) that catalyze the initial, rate-limiting step of mitochondrial fatty acid ␤-oxidation, with distinct but overlapping fatty acyl chain-length specificities (1, 2). In addition to VLCAD, which has optimal chain length specificity for fatty acyl-CoAs having 16 carbons in length, there are long-, medium-, and short-chain acyl-CoA dehydrogenases (LCAD, MCAD, and SCAD), which are most active with 14, 8, and 4 carbon substrates, respectively (3, 4). In addition, acyl-CoA dehydrogenase 9 (ACAD-9) is most active with unsaturated long-chain acyl-CoAs (5). The ACAD family also includes four members involved in amino acid metabolic pathways: isobutyryl-CoA dehydrogenase (IBD) in valine metabolism, isovaleryl-CoA dehydrogenase (IVD) in leucine metabolism, glutaryl-CoA dehydrogenase (GCAD) in lysine and tryptophan metabolism, and short-branched chain acylCoA dehydrogenase (SBCAD) in isoleucine metabolism. Fatty acyl-CoAs are oxidized to the corresponding trans-2,3-enoylCoA products with a concurrent reduction of the enzymebound FAD cofactor (6). Electron transfer flavoprotein (ETF) reoxidizes the reduced flavin and transfers reducing equivalents to the main mitochondrial respiratory chain through the enzyme ETF-ubiquinone oxidoreductase (7). Unlike other ACADs, which are soluble homotetramers with 45-kDa subunits, mature VLCAD and A...

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Section: Resultsmentioning

confidence: 99%

Structural Basis for Substrate Fatty Acyl Chain Specificity

McAndrew

Wang

Mohsen

et al. 2008

Journal of Biological Chemistry

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“…It is a wellestablished fact that reduced MCADH with bound product (or product analogs) is reoxidized by molecular oxygen much slower than the reduced enzyme without the bound ligand (Beinert, 1963;Wang & Thorpe, 1991;Trieval et al, 1995). In their studies with redox-inactive analogs, Thorpe and coworkers (Trievel et al, 1995) have shown that the ligands which lack the carbonyl oxygen (e.g., thioether) are much less effective (at least 2 orders of magnitude) in protecting the reduced enzyme flavin toward molecular oxygen. This could be explained as following: First, the thioether ligand would bind less tightly to the enzyme than the corresponding thioester, due to lack of the two hydrogen bonds between the carbonyl oxygen to the flavin ribityl 2′-OH and to the main chain amide nitrogen of amino acid 376.…”

Section: Discussionmentioning

confidence: 99%

Crystal Structures of the Wild Type and the Glu376Gly/Thr255Glu Mutant of Human Medium-Chain Acyl-CoA Dehydrogenase: Influence of the Location of the Catalytic Base on Substrate Specificity

et al. 1996

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Crystal structures of the wild type human medium-chain acyl-CoA dehydrogenase (MCADH) and a double mutant in which its active center base-arrangement has been altered to that of long chain acyl-CoA dehydrogenase (LCADH), Glu376Gly/Thr255Glu, have been determined by X-ray crystallography at 2.75 and 2.4 Å resolution, respectively. The catalytic base responsible for the R-proton abstraction from the thioester substrate is Glu376 in MCADH, while that in LCADH is Glu255 (MCADH numbering), located over 100 residues away in its primary amino acid sequence. The structures of the mutant complexed with C8-, C12, and C14-CoA have also been determined. The human enzyme structure is essentially the same as that of the pig enzyme. The structure of the mutant is unchanged upon ligand binding except for the conformations of a few side chains in the active site cavity. The substrate with chain length longer than C12 binds to the enzyme in multiple conformations at its ω-end. Glu255 has two conformations, "active" and "resting" forms, with the latter apparently stabilized by forming a hydrogen bond with Glu99. Both the direction in which Glu255 approaches the C R atom of the substrate and the distance between the Glu255 carboxylate and the C R atom are different from those of Glu376; these factors are responsible for the intrinsic differences in the kinetic properties as well as the substrate specificity. Solvent accessible space at the "midsection" of the active site cavity, where the C R -C bond of the thioester substrate and the isoalloxazine ring of the FAD are located, is larger in the mutant than in the wild type enzyme, implying greater O 2 accessibility in the mutant which might account for the higher oxygen reactivity.Acyl-CoA dehydrogenases are a family of flavoenzymes that catalyze the R, -dehydrogenation of acyl-CoA thioesters to the corresponding trans-enoyl-CoA with transfer of reducing equivalents to electron transfer flavoprotein (Beinert, 1963). In mammalian mitochondria, four straight-chain specific acyl-CoA dehydrogenases [short (SCADH)-, medium (MCADH)-, long (LCADH)-, and very long chain (VLCAD) acyl-CoA dehydrogenase] involved in fatty acid catabolism (Beinert, 1963;Aoyama et al., 1994) and three branched-chain specific acyl-CoA dehydrogenases (isovaleryl-, 2-methylbutyryl-, and glutaryl-CoA-DH) in amino acid metabolism have been described (Tanaka & Indo, 1992;Lenich & Goodman, 1986). Among them, MCADH is the most studied: the three dimensional structure of pig MCADH has been determined with and without various substrates and inhibitors (Kim et al., 1993 and its catalytic residue has been identified (Powell & Thorpe, 1988) and confirmed by site-specific mutagenesis (Bross, et al, 1990) and by the three dimensional structures of enzyme-substrate complex (Kim et al., 1993). All seven acyl-CoA dehydrogenases have been cloned, sequenced, and over-expressed in bacterial systems. Of the 27 known amino acid sequences of acylCoA dehydrogenases from both mammalian and bacterial sources available, the catal...

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“…The 3-thia sulfur of 3S-C 8 -CoA lowers the pK a of the adjacent R-proton by approximately 5 pK units compared to n-octanoyl-CoA (16), for which a pK a ≈21 has been estimated (8,9). Hence, a pK a ≈16 for the RC-H of free 3S-C 8 CoA appears to be an educated guess.…”

Section: Formation Of Anionic Ligands Upon Binding To Mcadhmentioning

confidence: 99%

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

et al. 1998

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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 ...

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Role of the Carbonyl Group in Thioester Chain Length Recognition by the Medium Chain Acyl-CoA Dehydrogenase

Cited by 35 publications

References 46 publications

Structural Basis for Substrate Fatty Acyl Chain Specificity

Structural Basis for Substrate Fatty Acyl Chain Specificity

Crystal Structures of the Wild Type and the Glu376Gly/Thr255Glu Mutant of Human Medium-Chain Acyl-CoA Dehydrogenase: Influence of the Location of the Catalytic Base on Substrate Specificity

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

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