title: Kinetic and spectral properties of isovaleryl-CoA dehydrogenase and interaction with ligands.
abstract: Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and the transfer of electrons to the electron transfer flavoprotein (ETF). Recombinant human IVD purifies with bound CoA-persulfide. A modified purification protocol was developed to isolate IVD without bound CoA-persulfide and to protect the protein thiols from oxidation. The CoA-persulfide-free IVD specific activity was 112.5 μmol porcine ETF min(-)(1) mg(-)(1), which was ∼20-fold higher than that of its CoA-persulfide bound form. The Km and catalytic efficiency (kcat/Km) for isovaleryl-CoA were 1.0 μM and 4.3 × 10(6) M(-1) s(-1) per monomer, respectively, and its Km for ETF was 2.0 μM. Anaerobic titration of isovaleryl-CoA into an IVD solution resulted in a stable blue complex with increased absorbance at 310 nm, decreased absorbance at 373 and 447 nm, and the appearance of the charge transfer complex band at 584 nm. The apparent dissociation constant (KDapp) determined spectrally for isovaleryl-CoA was 0.54 μM. Isovaleryl-CoA, acetoacetyl-CoA, methylenecyclopropyl-acetyl-CoA, and ETF induced CD spectral changes at the 250-500 nm region while isobutyryl-CoA did not, suggesting conformational changes occur at the flavin ring that are ligand specific. Replacement of the IVD Trp166 with a Phe did not block IVD interaction with ETF, indicating that its indole ring is not essential for electron transfer to ETF. A twelve amino acid synthetic peptide that matches the sequence of the ETF docking peptide competitively inhibited the enzyme reaction when ETF was used as the electron acceptor with a Ki of 1.5 mM.
authors.affiliation: Department of Pediatrics, School of Medicine, Children's Hospital of Pittsburgh, University of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA 15224, USA. Electronic address: aam27@pitt.edu.
authors.forename: Al-Walid A
authors.initials: AW
authors.lastname: Mohsen
authors.affiliation: Department of Pediatrics, School of Medicine, Children's Hospital of Pittsburgh, University of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA 15224, USA; Department of Human Genetics, Graduate School of Public Health, Pittsburgh, PA 15213, USA.
authors.forename: Jerry
authors.initials: J
authors.lastname: Vockley
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism; D002942:Circular Dichroism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D050769:Isovaleryl-CoA Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D013440:Sulfides / Q000378:metabolism
chemical_list: D000214:Acyl Coenzyme A; D008024:Ligands; D013440:Sulfides; C010667:acetoacetyl CoA; C010750:methylenecyclopropyl acetyl-CoA; D000105:Acetyl Coenzyme A; D050769:Isovaleryl-CoA Dehydrogenase
keywords: Acyl-CoA dehydrogenase; Electron-transferring flavoprotein