Influence of excision of a methylene group from Glu-376 (Glu376-->Asp mutation) in the medium chain acyl-CoA dehydrogenase-catalyzed reaction.

The human liver medium chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction proceeds via abstraction of an alpha-proton from the acyl-CoA substrates by the carboxyl group of Glu-376. By using the methods of site-directed mutagenesis, we replaced Glu-376 by Asp (E376D mutation), expressed the wild-type and mutant enzymes in Escherichia coli, purified them to homogeneity, and compared their kinetic properties. The steady-state kinetic data revealed that the E376D mutation impaired (by about 15-20-fold) the turnover rate of the enzyme as well as its inactivation by 2-octynoyl-CoA. There was no selective solvent deuterium isotope effect on enzyme catalysis. These results lead to the suggestion that the carboxyl group of Asp-376 does not serve as efficient catalytic base as the carboxyl group of Glu-376. The E376D mutation impaired the octanoyl-CoA-dependent reductive half-reaction such that the rate-limiting step of enzyme catalysis shifted from the product dissociation step (in the case of the wild-type enzyme) to the flavin reduction step, and abolished the previously noted kinetic and thermodynamic correspondences between the octanoyl-CoA-dependent reductive half-reaction and the enzyme-octenoyl-CoA interaction [Kumar, N. R., and Srivastava, D. K. (1994) Biochemistry 33, 8833-8841]. Arguments are presented that the Glu-376-->Asp mutation results in uncoupling between the proton transfer and protein conformational change steps during enzyme catalysis.