Mechanism-based inhibitor discrimination in the acyl-CoA dehydrogenases.
The catalytically essential glutamate base in the acyl-CoA dehydrogenase family is found either on the loop between J and K helices (e.g., in short-chain, medium-chain, and glutaryl-CoA dehydrogenases) or on the G helix (long-chain and isovaleryl-CoA dehydrogenases). While active-site bases at either position are functionally equivalent with respect to alpha-proton abstraction, reactions that require removal of a gamma-proton show marked differences between the two enzyme classes. Thus short-chain, medium-chain, and glutaryl-CoA dehydrogenase are rapidly inactivated by 2-pentynoyl-CoA with abstraction of a gamma-proton, whereas isovaleryl-CoA dehydrogenase is not significantly inhibited. This resistance is not due to weak binding: the complex between isovaleryl-CoA dehydrogenase and 2-pentynoyl-CoA shows a Kd of 1.8 microM at pH 7.6. Migration of the catalytic base to the loop between J and K helices (using the Glu254Gly/Ala375Glu double mutant) makes isovaleryl-CoA dehydrogenase sensitive to irreversible inhibition by 2-pentynoyl-CoA. Molecular modeling suggests that this mutation brings the catalytic base close enough to abstract a gamma-proton from the bound inhibitor. Experiments with two mechanism-based inactivators that target the FAD of the medium- and short-chain acyl-CoA dehydrogenases support this conclusion. 3-Methyl-3-butenoyl-CoA requires activation by alpha-proton abstraction and rapidly yields a reduced flavin adduct with wild-type isovaleryl-CoA dehydrogenase. In contrast, the isomeric 3-methyl-2-butenoyl-CoA is inert toward this enzyme because it cannot be activated by gamma-proton abstraction. Molecular modeling supports these observations. This unusual selectivity toward mechanism-based inactivators provides additional discrimination between members of the acyl-CoA dehydrogenase family.
Schaller
R A
RA
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
Mohsen
A W
AW
Vockley
J
J
Thorpe
C
C
D044942:Acyl-CoA Dehydrogenase, Long-Chain / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D003065:Coenzyme A / Q000378:metabolism; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008930:Mitochondria, Liver / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010084:Oxidation-Reduction; D011485:Protein Binding; D013552:Swine
D004791:Enzyme Inhibitors; D044942:Acyl-CoA Dehydrogenase, Long-Chain; D003065:Coenzyme A