Isothermal titration microcalorimetric studies for the binding of octenoyl-CoA to medium chain acyl-CoA dehydrogenase

Biochemistry
D K SrivastavaK L Peterson

Abstract

We investigated the binding of octenoyl-CoA to pig kidney medium chain acyl-CoA dehydrogenase (MCAD) by isothermal titration microcalorimetry under a variety of experimental conditions. At 25 degrees C in 50 mM phosphate buffer at pH 7.6 (ionic strength of 175 mM), the binding is characterized by the stoichiometry (n) of 0.89 mole of octenoyl-CoA/(mole of MCAD subunit), delta G = -8.75 kcal/mol, delta H = -10.3 kcal/mol, and delta S = -5.3 cal mol(-1) K(-1), suggesting that formation of MCAD-octenoyl-CoA is enthalpically driven. By employing buffers with various ionization enthalpies, we discerned that formation of the MCAD-octenoyl-CoA complex, at pH 7.6, accompanies abstraction (consumption) of 0.52 +/- 0.15 proton/(MCAD subunit) from the buffer media. We studied the effects of pH, ionic strength, and temperature on the thermodynamics of MCAD-octenoyl-CoA interaction. Whereas the ionic strength does not significantly influence the above interaction, the pH of the buffer media exhibits a pronounced effect. The pH dependence of the association constant of MCAD +octenoyl-CoA <==> MCAD-octenoyl-CoA yields a pKa for the free enzyme of 6.2. Among thermodynamic parameters, whereas delta G remains invariant as a function of temperatu...Continue Reading

References

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Citations

Jul 14, 2001·Biochimica Et Biophysica Acta·E Ortiz-SalmerónL García-Fuentes
Aug 8, 2001·European Journal of Biochemistry·E Ortiz-SalmerónL García-Fuentes
Mar 5, 2016·Plant Physiology and Biochemistry : PPB·Jose A Aznar-MorenoJoaquín J Salas
Sep 29, 2004·Biochimica Et Biophysica Acta·Irene Domínguez-PérezLuis García-Fuentes
May 14, 2003·Biomacromolecules·Uracha RungsardthongSnjezana Stolnik
Jun 28, 2001·Journal of the American Chemical Society·M A FazalK R Rodgers
Jul 10, 2003·Journal of the American Chemical Society·Mickael MichaudEric Peyrin

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