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.
Peterson
K L
KL
Biochemistry Department, North Dakota State University, Fargo 58105, USA.
Galitz
D S
DS
Srivastava
D K
DK
D000214:Acyl Coenzyme A / Q000378:metabolism; D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics*; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics*; D006801:Humans; D006838:Hydrocarbons; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008697:Methane / Q000031:analogs & derivatives* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013552:Swine
D000214:Acyl Coenzyme A; D006838:Hydrocarbons; C030011:carbene; D001224:Aspartic Acid; D018698:Glutamic Acid; C058947:2-octynoyl-coenzyme A; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase; D008697:Methane