Arginine 387 of human isovaleryl-CoA dehydrogenase plays a crucial role in substrate/product binding.
Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric flavoenzyme, which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and transfers electrons to the electron-transferring flavoprotein, and is a member of the acyl-CoA dehydrogenase (ACD) enzyme family. Human IVD crystal structure with a bound substrate analogue shows the guanidino group of Arg387, a conserved residue among other members of the ACD enzyme family, juxtaposed to a phosphate oxygen of the 4'-phosphopantothiene moiety of the substrate analogue. Site-directed mutagenesis was used to investigate the role of Arg387 in substrate binding and enzyme function. Replacing this residue with Lys, Ala, Gln, or Glu resulted in stable proteins. Spectrophotometric substrate binding assays indicated that the Arg387Lys mutant was able to form the charge-transfer complex intermediate with similar efficiency to wild type, while the rest of the mutants were significantly less able to properly form this intermediate. However, the Km of the isovaleryl-CoA for the Arg387Lys mutant was 20.3 compared to 1.5 microM for the wild type. The Km for the rest of the mutants were 75.6, 195, and 550 microM, respectively. The catalytic efficiency per mole of FAD was 20.3, 3.3, 2.0, and 0.34 for the mutants, respectively, compared to 260 microM(-1) x min(-1) for the wild type. These results substantiate the important role of Arg387 in anchoring the substrate, and are consistent with the hypothesis that residues distant from the active site are important for stabilizing the enzyme:substrate/product complex, and could play an important role in the mechanism of the enzyme-catalyzed reaction.
Volchenboum
S L
SL
Department of Medical Genetics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
Mohsen
A W
AW
Kim
J J
JJ
Vockley
J
J
D000214:Acyl Coenzyme A / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004735:Energy Transfer / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D006801:Humans; D050769:Isovaleryl-CoA Dehydrogenase; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity / Q000235:genetics / Q000502:physiology*
D000214:Acyl Coenzyme A; D011994:Recombinant Proteins; C017447:isovaleryl-coenzyme A; C033618:tiglyl-coenzyme A; D001120:Arginine; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050769:Isovaleryl-CoA Dehydrogenase; D008239:Lysine