A novel approach to the characterization of substrate specificity in short/branched chain Acyl-CoA dehydrogenase.
Rat and human short/branched chain acyl-CoA dehydrogenases exhibit key differences in substrate specificity despite an overall amino acid identity of 85% between them. Rat short/branched chain acyl-CoA dehydrogenases (SBCAD) are more active toward substrates with longer carbon side chains than human SBCAD, whereas the human enzyme utilizes substrates with longer primary carbon chains. The mechanism underlying this difference in substrate specificity was investigated with a novel surface plasmon resonance assay combined with absorbance and circular dichroism spectroscopy, and kinetics analysis of wild type SBCADs and mutants with altered amino acid residues in the substrate binding pocket. Results show that a relatively few amino acid residues are critical for determining the difference in substrate specificity seen between the human and rat enzymes and that alteration of these residues influences different portions of the enzyme mechanism. Molecular modeling of the SBCAD structure suggests that position 104 at the bottom of the substrate binding pocket is important in determining the length of the primary carbon chain that can be accommodated. Conformational changes caused by alteration of residues at positions 105 and 177 directly affect the rate of electron transfer in the dehydrogenation reactions, and are likely transmitted from the bottom of the substrate binding pocket to beta-sheet 3. Differences between the rat and human enzyme at positions 383, 222, and 220 alter substrate specificity without affecting substrate binding. Modeling predicts that these residues combine to determine the distance between the flavin ring of FAD and the catalytic base, without changing the opening of the substrate binding pocket.
He
Miao
M
Department of Medical Genetics, Mayo Clinic, Rochester, Minnesota 55905, USA.
Burghardt
Thomas P
TP
Vockley
Jerry
J
D000214:Acyl Coenzyme A / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
D000214:Acyl Coenzyme A; C044181:hexanoyl-coenzyme A; D044925:Oxidoreductases Acting on CH-CH Group Donors; C038808:2-methylacyl-CoA dehydrogenase