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J Biol Chem. 1980 Oct 10;255(19):9093-7.

The deuterium isotope effect upon the reaction of fatty acyl-CoA dehydrogenase and butyryl-CoA.


Reduction of the oxidized FAD at the active site of porcine liver fatty acyl-CoA dehydrogenase by butyryl-CoA results in bleaching of 30 to 60% of the 450 nm absorbance of flavin and in the production of a new absorbance band at 565 nm. The wavelength of the maximum absorbance of this new band (lambda max) is dependent on the chemical nature of the substrate, e.g. this band occurs at 645 nm when beta-2-furylpropionyl-CoA (a pseudosubstrate) reacts with enzyme. Since lambda max for this band is substrate-dependent, the band is most likely the result of charge transfer complex formation between oxidized fatty acyl-CoA, and the reduced flavin of the enzyme. The rate profile for the reaction of butyryl-CoA and enzyme is biphasic at 450 nm but consists of a single exponential process at 565 nm. The rate constant for reaction at 565 nm is approximately 12 s-1 ((butyryl-CoA) = 2.5 x 10(-5) M, pH 8.6), and the 450 nm rate profile can be fit to a rate equation for two sequential reactions of rate constant 12 s-1 and 3.4 s-1, the amount of flavin reduction in each kinetic step being approximately 50%. The deuterium isotope effect measured on each step of the biphasic time course of the 450 nm reaction is very large, in the range kH/kD = 30 to 50. The rate profile at 565 nm for perdeuterobutyryl-CoA is markedly different than that for the protiobutyryl-CoA in that it is biphasic. It appears that two rate processes have been separated by virtue of different isotope effects; the first process shows kH/kD = 2 while the second shows kH/kD = 50. The data are interpreted in terms of a mechanism involving an obligatory charge transfer complex.

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