Reductive half-reaction in medium-chain acyl-CoA dehydrogenase: modulation of internal equilibrium by carboxymethylation of a specific methionine residue.
Pig kidney medium-chain acyl-CoA dehydrogenase is specifically alkylated at a methionine residue by treatment with iodoacetate at pH 6.6. This residue corresponds to Met249 in the human medium-chain acyl-CoA dehydrogenase sequence [Kelly, D. P., Kim, J. J., Billadello, J. J., Hainline, B. E., Chu, T. W., & Strauss, A. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4068-4072]. The S-carboxymethylated dehydrogenase shows a drastically lowered affinity for octanoyl-CoA (from submicromolar to 65 microM), but retains about 23% of the maximal activity of the native enzyme. In addition, alkylation perturbs the internal redox equilibrium: E.FADox.octanoyl-CoA K2 in equilibrium with E.FAD2e.octenoyl-CoA K2 ranges from about 9 for the native enzyme to about 0.2 for the homogeneously modified protein. This effect is not due to a significant change in the redox potential of the free enzyme upon alkylation. Rather, carboxymethylation weakens the preferential binding of enoyl-CoA product to the reduced enzyme (K3) compared to octanoyl-CoA binding to the oxidized dehydrogenase (K1) that is required to pull the substrate thermodynamically uphill. Thus, the ratio of dissociation constants, K1/K3, decreases from about 15,000 for the native enzyme to only 330 upon carboxymethylation of Met249. Binding studies with a variety of acyl-CoA analogues and manipulation of enzyme redox potentials by substitution of the natural prosthetic group by 8-Cl-FAD confirm the thermodynamic effects of alkylation.
Cummings
J G
JG
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Lau
S M
SM
Powell
P J
PJ
Thorpe
C
C
D000214:Acyl Coenzyme A / Q000378:metabolism; D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004734:Energy Metabolism; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007668:Kidney / Q000201:enzymology; D046911:Macromolecular Substances; D008715:Methionine; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments; D017385:Sequence Homology; D013329:Structure-Activity Relationship; D013552:Swine
D000214:Acyl Coenzyme A; D007461:Iodoacetates; D046911:Macromolecular Substances; D010446:Peptide Fragments; C044771:3,4-pentadienoyl-coenzyme A; D008715:Methionine; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase; D019807:Iodoacetic Acid