Product Binding Modulates the Thermodynamic Properties of a Megasphaera elsdenii Short-Chain Acyl-CoA Dehydrogenase Active-Site Mutant

Donald F. Becker, James A. Fuchs, Marian T. Stankovich

Research output: Contribution to journalArticle

18 Scopus citations

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 Glu367GlnSCAD 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.

Original languageEnglish (US)
Pages (from-to)7082-7087
Number of pages6
JournalBiochemistry
Volume33
Issue number23
DOIs
StatePublished - Jun 1 1994

ASJC Scopus subject areas

  • Biochemistry

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