2-Octynoyl coenzyme A is a mechanism-based inhibitor of pig kidney medium-chain acyl coenzyme A dehydrogenase: isolation of the target peptide

Powell, Patricia J.; Thorpe, Colin

doi:10.1021/bi00421a008

Cited by 89 publications

(110 citation statements)

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“…Thus, the microscopic pK a of the substrate RC-H bond should be lowered from an estimated value of ≈20 (17-19) toward ≈9. In the substrate analogue 3S-C 8 CoA, the use of which was introduced earlier by Thorpe's group (20), we showed that the microscopic RC-H pK a is lowered from ≈16 in the free state to ≈5 on binding to the active center of hwtMCADH (14). Analogously, with the "chromogenic transition-state analogue" p-nitrophenylacetyl-CoA, the same pK a is shifted from ≈13.6 to ≈5 (14).…”

mentioning

confidence: 70%

“…Diffraction quality crystals were obtained from crystallization drops (8 µL), each containing 52.5 µg of the enzyme in 50 mM Tris acetate, 50 mM NaCl, 4.5% poly(ethylene glycol) (PEG) 8000, that had been equilibrated against a solution containing 10% PEG 8000 and 0.1 M NaCl. Crystals of the complex with C 8 CoA were obtained by soaking preformed holoenzyme crystals in mother liquor containing 1.4 mM C 8 CoA for 18 h. No special precautions were taken to keep the crystals in anaerobic conditions. X-ray data sets were collected at 4°C, using an R-AXIS IIC image-plate system with CuKR radiation generated from a Rigaku RU200 rotating anode generator.…”

Section: Methodsmentioning

confidence: 99%

“…The microscopic pK a of Glu376sCOO -recently has been determined to be ≈6 in uncomplexed enzyme (14). It is increased to [8][9] in the presence of ligands (4,14), and is probably still higher with good substrates such as C 8 CoA (14)(15)(16). Thus, the microscopic pK a of the substrate RC-H bond should be lowered from an estimated value of ≈20 (17-19) toward ≈9.…”

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confidence: 99%

“…To help resolve this point and to assess the importance of the corresponding H-bond, we set out to prepare enzyme reconstituted with both 2′-and 3′-deoxy-FAD analogues. However, as we refined the structure to a higher resolution (2.4 Å), it became evident that the carbonyl group of C 8 CoA was hydrogen bonded to 2′-OH of the ribityl chain of FAD as well as to the main-chain amide nitrogen atom of Glu376 (26) (distances 2.8 and 3.0 Å, respectively).…”

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confidence: 99%

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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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confidence: 70%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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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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“…Another distinction between the action of Glu376 and of Glu255 lies in their interactions with 2-octynoyl-CoA. MCADH is irreversibly inhibited by 2-octynoyl-CoA by covalent modification at Glu376 (Powell & Thorpe, 1988), but neither beef liver LCADH (Ankele et al, 1991) nor MLCADH (Nandy et al, 1996) is covalently modified by the inhibitor. Although 2-octynoyl-CoA is shown to be a mechanism-based inhibitor involving rate-limiting removal of one of the C γ protons (Lau et al, 1988), the exact site of the covalent linkage with the glutamate on the inhibitor is not known at present.…”

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 biochemistry of enols

Richard

1990

PATAI'S Chemistry of Functional Groups

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2-Octynoyl coenzyme A is a mechanism-based inhibitor of pig kidney medium-chain acyl coenzyme A dehydrogenase: isolation of the target peptide

Cited by 89 publications

References 33 publications

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

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

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

The biochemistry of enols

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