title: Product binding modulates the thermodynamic properties of a Megasphaera elsdenii short-chain acyl-CoA dehydrogenase active-site mutant.
abstract: Previous work has shown that the redox properties of Megasphaera elsdenii short-chain acyl-CoA dehydrogenase (SCAD) are specifically modulated upon the binding of the substrate/product couple, allowing the reaction to proceed thermodynamically [Stankovich, M.T., & Soltysik, S. (1987) Biochemistry 26, 2627-2632]. The focus of this study on the Glu367Gln SCAD mutant protein is to gain an understanding of this phenomenon. The active-site mutant Glu367Gln SCAD inactivates the reductive and oxidative pathways and allows the effects of substrate (butyryl-CoA) and product (crotonyl-CoA) binding on the redox properties of the Glu367Gln SCAD mutant protein to be determined separately. Red anionic semiquinone was found to be thermodynamically stabilized in coulometric/potentiometric reductions of both butyryl-CoA- and crotonyl-CoA-complexed Glu367Gln SCAD. Reduction potential measurements showed that butyryl-CoA binding has little effect on the reduction potential of Glu367Gln SCAD. Crotonyl-CoA complexation, however, shifted the reduction potential of the Glu367Gln SCAD mutant protein by 30 mV in the positive direction. This modulation is similar to the 60-mV positive shift observed in native M. elsdenii SCAD upon complexation with the substrate/product couple [Stankovich, M.T., & Soltysik, S. (1987) Biochemistry 26, 2627-2632]. Thus, product binding and not substrate binding, thermodynamically regulates M. elsdenii SCAD. We propose that this observation is best explained by assuming that the product resembles an intermediate in the catalytic mechanism that is responsible for facilitating isopotential electron transfer from the substrate to the enzyme.
authors.affiliation: Chemistry and Biochemistry Department, University of Minnesota, Minneapolis 55455.
authors.forename: D F
authors.initials: DF
authors.lastname: Becker
authors.forename: J A
authors.initials: JA
authors.lastname: Fuchs
authors.forename: M T
authors.initials: MT
authors.lastname: Stankovich
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004563:Electrochemistry; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D013816:Thermodynamics
chemical_list: D000214:Acyl Coenzyme A; C024343:butyryl-coenzyme A; C010701:crotonyl-coenzyme A; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase