The binding of substrate/product or transition-state intermediates modifies the properties of medium-chain fatty acyl-CoA dehydrogenase (MCAD) by causing the redox potential to shift positive and the oxygen reactivity to slow by 3000-fold. Two ligands, identified as being the most effective in slowing oxygen reactivity, were 2-azaoctanoyl-CoA and 3-thiaoctanoyl-CoA [Wang, R., & Thorpe, C. (1991) Biochemistry 30, 7895-7901]. We have measured the potential shifts caused by the binding of both ligands to determine which is most similar to the potential shift caused by substrate/product mixture, the assumption being that the best transition-state structural intermediate would give the potential shift most similar to that of substrate/product [Lenn, N.D., Stankovich, M.T., & Liu, H. (1990) Biochemistry 29, 10594-10602]. Both ligands shifted the potential positive, but the shift caused by 2-azaoctanoyl-CoA was 65% that of substrate/product, while 3-thiaoctanoyl-CoA was only 20% of that value. This positive shift is proposed to be caused by a resonance form stabilized by the interaction of the catalytically essential carbonyl of the acyl-CoA with two hydrogen bonds from the enzyme, which induces a partial negative charge on the carbonyl and a partial positive charge on carbon 2 of the ligand and carbon 3 of the substrate/product couple. The X-ray structure shows that carbons 2 and 3 of the substrate/product overlap the diazadiene portion of the flavin ring [Kim, J.-J. P., Wang, M., & Paschke, R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7523-7527].(ABSTRACT TRUNCATED AT 250 WORDS)
The modulation of the electron-transfer properties of human medium-chain acyl-CoA dehydrogenase (hwtMCADH) has been studied using wild-type and site-directed mutants by determining their midpoint potentials at various pH values and estimating the involved pKs. The mutants used were E376D, in which the negative charge is retained; E376Q, in which one negative charge (pK a ≈ 6.0) is removed from the active center; E99G, in which a different negative charge (pK a ≈ 7.3) also is affected; and E376H (pK a ≈ 9.3) in which a positive charge is present. E m for hwtMCADH at pH 7.6 is -0.114 V. Results for the site-directed mutants indicate that loss of a negative charge in the active site causes a +0.033 V potential shift. This is consistent with the assumption that electrostatic interactions (as in the case of flavodoxins) and specific charges are important in the modulation of the electron-transfer properties of this class of dehydrogenases. Specifically, these charge interactions appear to correlate with the positive E m shift observed upon binding of substrate/product couple to MCADH [Lenn, N. D., Stankovich, M. T., and Liu, H. (1990) Biochemistry 29, 3709-3715], which coincides with a pK increase of Glu376-COOH from ∼6 to 8-9 [Rudik, I., Ghisla, S., and Thorpe, C. (1998) Biochemistry 37, 8437-8445]. From the pH dependence of the midpoint potentials of hwtMCADH two mechanistically important ionizations are estimated. The pK a value of ∼6.0 is assigned to the catalytic base, Glu376-COOH, in the oxidized enzyme based on comparison with the pH behavior of the E376H mutant, it thus coincides with the pK value recently estimated [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The pK a of ∼7.1 is assigned to Glu376-COOH in reduced hwtMCADH. Comparable values for these pK a s for Glu376-COOH in pig kidney MCADH are pK ox ) 6.5 and pK red ) 7.9. The E m measured for K304E-MCADH (a major mutant resulting in a deficiency syndrome) is essentially identical to that of hwtMCADH, indicating that the disordered enzyme has an intact active site.Mitochondrial fatty acid oxidation is a major energy source for mammals (1). The initial and key enzymes in -oxidation are the acyl-CoA dehydrogenases, members of a broad family, which catalyze the dehydrogenation of fatty acids with chain lengths of 4 up to >18 carbon atoms. Mediumchain acyl-CoA dehydrogenase (MCADH), with an optimum activity for C 8 -substrates, is the best studied member of this family. The reaction it catalyzes occurs formally in two steps as shown in eqs 1 and 2 (2, 3):where E ) MCADH, SH 2 ) reduced substrate, P ) product, ETF ) electron transferring flavoprotein, and its (•) semiquinone form. 1 The reductive half-reaction proceeds via the abstraction of both a proton and a hydride ion from the R-and -carbons of the thioester-CoA substrate, respectively, (4). Glu376-COO -is the proton abstracting base (5, 6). The carbonyl oxygen of the thioester is involved in two essential hydrogen bonds, one to the ribityl 2-hydroxyl group...
The national interest in science, technology, engineering and mathematics (STEM) has called attention to P-12 education, the STEM pipeline. Education of teachers is a primary influence on the education of children in the classroom. While high school (and often middle school) teachers are versed in the content of a particular aspect of STEM (e.g. Mathematics or Chemistry), elementary teachers, on the other end of the pipeline, are educated as generalists, with a primary goal of setting the foundations for future learning.In 2004, a team of STEM and education faculty at St. Catherine University (SCU) was called together, united by their interest in improving STEM education for all students at SCU, particularly women. Combining the content expertise of the biology, chemistry, physics/engineering, and mathematics departments with the methods expertise of the education department, the team designed courses that made STEM concepts more engaging and relevant to students. In 2010, the STEM Certificate was solidified and required of all elementary education students. It is comprised of three interdisciplinary, team-taught, lab based courses that are open to all undergraduate majors at the institution. Each course is centered on one discipline (i.e. biology, chemistry, or engineering/physics). Chemistry of Life is the chemistry-focused course. The course was designed to include a capstone project. As an introduction to materials science, nanoscience was selected as the theme for the projects. The topic allowed for socially relevant and also highly interdisciplinary projects. Students working in teams of three or four, designed projects, determined how to measure and obtain data, and analyzed and interpreted results. A content and confidence assessment given to students before and after the projects showed an increase in both their understanding of nanomaterials and their confidence in conducting a nanoscience project.
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