Mechanism-Based Inhibitor Discrimination in the Acyl-CoA Dehydrogenases
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- 作者：Ruth A. Schaller ; Al-Walid A. Mohsen ; Jerry Vockley ; and Colin Thorpe
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：June 24, 1997
- 年：1997
- 卷：36
- 期：25
- 页码：7761 - 7768
- 全文大小：205K
- 年卷期：v.36,no.25(June 24, 1997)
- ISSN：1520-4995
文摘
The catalytically essential glutamate base in theacyl-CoA dehydrogenase family is foundeither on the loop between J and K helices (e.g., in short-chain,medium-chain, and glutaryl-CoAdehydrogenases) or on the G helix (long-chain and isovaleryl-CoAdehydrogenases). While active-sitebases at either position are functionally equivalent with respect to
-proton abstraction, reactions thatrequire removal of a 
-proton show marked differences between the twoenzyme classes. Thus short-chain, medium-chain, and glutaryl-CoA dehydrogenase are rapidlyinactivated by 2-pentynoyl-CoA withabstraction of a -proton, whereas isovaleryl-CoA dehydrogenase isnot significantly inhibited. Thisresistance is not due to weak binding: the complex betweenisovaleryl-CoA dehydrogenase and2-pentynoyl-CoA shows a Kd of 1.8 
M at pH7.6. Migration of the catalytic base to the loopbetweenJ and K helices (using the Glu254Gly/Ala375Glu double mutant) makesisovaleryl-CoA dehydrogenasesensitive to irreversible inhibition by 2-pentynoyl-CoA. Molecularmodeling suggests that this mutationbrings the catalytic base close enough to abstract a -proton fromthe bound inhibitor. Experiments withtwo mechanism-based inactivators that target the FAD of the medium- andshort-chain acyl-CoAdehydrogenases support this conclusion. 3-Methyl-3-butenoyl-CoArequires activation by -protonabstraction and rapidly yields a reduced flavin adduct with wild-typeisovaleryl-CoA dehydrogenase. Incontrast, the isomeric 3-methyl-2-butenoyl-CoA is inert toward thisenzyme because it cannot be activatedby -proton abstraction. Molecular modeling supports theseobservations. This unusual selectivity towardmechanism-based inactivators provides additional discrimination betweenmembers of the acyl-CoAdehydrogenase family.
-proton abstraction, reactions thatrequire removal of a -proton show marked differences between the twoenzyme classes. Thus short-chain, medium-chain, and glutaryl-CoA dehydrogenase are rapidlyinactivated by 2-pentynoyl-CoA withabstraction of a -proton, whereas isovaleryl-CoA dehydrogenase isnot significantly inhibited. Thisresistance is not due to weak binding: the complex betweenisovaleryl-CoA dehydrogenase and2-pentynoyl-CoA shows a Kd of 1.8 M at pH7.6. Migration of the catalytic base to the loopbetweenJ and K helices (using the Glu254Gly/Ala375Glu double mutant) makesisovaleryl-CoA dehydrogenasesensitive to irreversible inhibition by 2-pentynoyl-CoA. Molecularmodeling suggests that this mutationbrings the catalytic base close enough to abstract a -proton fromthe bound inhibitor. Experiments withtwo mechanism-based inactivators that target the FAD of the medium- andshort-chain acyl-CoAdehydrogenases support this conclusion. 3-Methyl-3-butenoyl-CoArequires activation by -protonabstraction and rapidly yields a reduced flavin adduct with wild-typeisovaleryl-CoA dehydrogenase. Incontrast, the isomeric 3-methyl-2-butenoyl-CoA is inert toward thisenzyme because it cannot be activatedby -proton abstraction. Molecular modeling supports theseobservations. This unusual selectivity towardmechanism-based inactivators provides additional discrimination betweenmembers of the acyl-CoAdehydrogenase family.