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Solvent Isotope Effects in Reactions of Human Medium-Chain Acyl-CoA Dehydrogenase Active Site Mutants

Solvent Isotope Effects in Reactions of Human Medium-Chain Acyl-CoA Dehydrogenase Active Site Mutants

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GRADINARU, Robert, Richard SCHOWEN, Sandro GHISLA, 2007. Solvent Isotope Effects in Reactions of Human Medium-Chain Acyl-CoA Dehydrogenase Active Site Mutants. In: Biochemistry. 46(9), pp. 2497-2509. ISSN 0006-2960. eISSN 1520-4995

@article{Gradinaru2007Solve-8345, title={Solvent Isotope Effects in Reactions of Human Medium-Chain Acyl-CoA Dehydrogenase Active Site Mutants}, year={2007}, doi={10.1021/bi0614582}, number={9}, volume={46}, issn={0006-2960}, journal={Biochemistry}, pages={2497--2509}, author={Gradinaru, Robert and Schowen, Richard 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/123456789/8345"> <dc:contributor>Ghisla, Sandro</dc:contributor> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:42:50Z</dc:date> <dc:rights>deposit-license</dc:rights> <dc:creator>Gradinaru, Robert</dc:creator> <dcterms:abstract xml:lang="eng">Glu376, the base involved in substrate αH+ abstraction at the active center of medium-chain acyl-CoA dehydrogenase (MCAD), has been mutated to Gln and Gly. The mutants are active; however, their rates of dehydrogenation are lowered by approximately 5 orders of magnitude. Binding of the substrate octanoyl-CoA to Glu376Gln-MCAD involves (at least) two steps. The ensuing dehydrogenation reaction that corresponds to reduction of the flavin cofactor also occurs in two phases. These are interpreted to consist of a first, reversible step, followed by a slower, practically irreversible one. For Glu376Gln-MCAD, the log of the rates of dehydrogenation increases linearly with pH (slope = 1) in the pH range of 6-10, suggesting HO- as a reactant. The rates of the same reactions in D2O have the same pD profile and reflect a solvent kinetic isotope effect (SKIE) of ≈8.5. Glu376Gln+Glu99Gly-MCAD (studied to assess the role of Glu99 also present at the bottom of the active center cavity) has activities and activity profiles similar to those of Glu376Gln-MCAD. This excludes Glu99 as the active center base for Glu376Gln-MCAD catalysis. Proton inventories for the two phases of the dehydrogenation reaction were investigated at 4 and 25°C. The inventories at 25°C reflect a SKIE of ≈4.5; the profiles are "bowl-shaped", in which a transition-state contribution predominates. The profiles for the 4°C reaction are very unusual. That for the first phase can be analyzed on a two-step model with one step (80% rate-limiting) having a conformational reorganization with an isotope effect of 90-100, from small isotope effects at many protein sites, and the other step (20% rate-limiting) having an inverse isotope effect of ca. 2, characteristic of the reaction of hydroxide ion as a base. For the second phase, only a contribution from many protein sites with a KIE of ≈4.5 is observed. The results are compatible with a very rigid active site framework that must undergo rearrangements for dehydrogenation to take place, and specifically to allow access of HO-, the reactant that must neutralize the H+ abstracted from the αC-H substrate. The large isotope effects are attributed to the changes in state of several H-bonds that occur during the process.</dcterms:abstract> <dcterms:rights rdf:resource="https://creativecommons.org/licenses/by-nc-nd/2.0/legalcode"/> <dcterms:title>Solvent Isotope Effects in Reactions of Human Medium-Chain Acyl-CoA Dehydrogenase Active Site Mutants</dcterms:title> <dc:format>application/pdf</dc:format> <dcterms:bibliographicCitation>First publ. in: Biochemistry ; 46 (2007), 9. - S. 2497-2509</dcterms:bibliographicCitation> <dc:creator>Ghisla, Sandro</dc:creator> <dc:creator>Schowen, Richard</dc:creator> <dc:contributor>Gradinaru, Robert</dc:contributor> <dc:language>eng</dc:language> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/8345"/> <dcterms:issued>2007</dcterms:issued> <dc:contributor>Schowen, Richard</dc:contributor> </rdf:Description> </rdf:RDF>

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