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

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

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VOCK, Petra, Stefan ENGST, Michael EDER, Sandro GHISLA, 1998. Substrate Activation by Acyl-CoA Dehydrogenases : Transition-State Stabilization and pKs of Involved Functional Groups. In: Biochemistry. 37(7), pp. 1848-1860. ISSN 0006-2960. eISSN 1520-4995

@article{Vock1998Subst-6835, title={Substrate Activation by Acyl-CoA Dehydrogenases : Transition-State Stabilization and pKs of Involved Functional Groups}, year={1998}, doi={10.1021/bi971827h}, number={7}, volume={37}, issn={0006-2960}, journal={Biochemistry}, pages={1848--1860}, author={Vock, Petra and Engst, Stefan and Eder, Michael and Ghisla, Sandro} }

<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/rdf/resource/123456789/6835"> <dcterms:rights rdf:resource="https://creativecommons.org/licenses/by-nc-nd/2.0/legalcode"/> <dcterms:title>Substrate Activation by Acyl-CoA Dehydrogenases : Transition-State Stabilization and pKs of Involved Functional Groups</dcterms:title> <dc:creator>Eder, Michael</dc:creator> <dc:creator>Ghisla, Sandro</dc:creator> <dc:contributor>Eder, Michael</dc:contributor> <dc:rights>deposit-license</dc:rights> <dcterms:bibliographicCitation>First publ. in: Biochemistry ; 37 (1998), 7. - S. 1848-1860</dcterms:bibliographicCitation> <dc:format>application/pdf</dc:format> <dcterms:abstract xml:lang="eng">The mechanism by which acyl-CoA dehydrogenases initiate catalysis was studied by using p-substituted phenylacetyl-CoAs (substituents -NO2, -CN, and CH3CO-), 3S-C8-, and 3'-dephospho-3S-C8CoA. These analogues lack a βC-H and cannot undergo α,β-dehydrogenation. Instead they deprotonate at αC-H at pH ≥ 14 to form delocalized carbanions having strong absorbancies in the near UV-visible spectrum. The pKas of the corresponding phenylacetone analogues were determined as ≈13.6 (-NO2), ≈14.5 (-CN), and ≈14.6 (CH3CO-). Upon binding to human wild-type medium-chain acyl-CoA dehydrogenase (MCADH), all analogues undergo αC-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 (Kd) of p-NO2-phenylacetyl-CoA (4NPA-CoA), 3S-C8-CoA, and 3'-dephospho-3S-C8CoA, four pKas at ≈5, ≈6, ≈7.3, and ≈8 were identified. They were assigned to the following ionizations: (a) pKa ≈5, ligand (L-H) in the MCADH~ligand complex; (b) pKa ≈6, Glu376-COOH in uncomplexed MCADH; (c) pKa ≈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 pKa ≈6 (b) is not significantly affected in the MCADH~4NPA-CoA complex, but it is increased by ≥1 pK unit in that with 3S-C8CoA and further in the presence of C8-CoA, the best substrate. The αC-H pKas of 4NPA-CoA, of 3S-C8-CoA, and of 3'-dephospho-3S-C8CoA in the complex with MCADH are ≈5, ≈5, and ≈6. Compared to those of the free species these pKa values are therefore lowered by 8 to ≥11 pH units (50 to ≥65 kJ mol-1) and are close to the pKa 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 pKa 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, αC-H abstraction is fast (kapp ≈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 pKas of ≈5 and ≈7.3, which correspond to those determined in static experiments.</dcterms:abstract> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/6835"/> <dc:creator>Vock, Petra</dc:creator> <dc:contributor>Vock, Petra</dc:contributor> <dc:contributor>Engst, Stefan</dc:contributor> <dc:language>eng</dc:language> <dc:contributor>Ghisla, Sandro</dc:contributor> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:29:31Z</dc:date> <dc:creator>Engst, Stefan</dc:creator> <dcterms:issued>1998</dcterms:issued> </rdf:Description> </rdf:RDF>

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