S-2-Bromo-Acyl-CoA Analogs Are Affinity Labels for the Medium-Chain Acyl-CoA Dehydrogenase from Pig-Kidney

Haeffner‐Gormley, Lorraine; Cummings, J G; Thorpe, Colin

doi:10.1006/abbi.1995.1191

Cited by 5 publications

(6 citation statements)

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“…For example, S ‐2‐bromohexanoyl‐CoA (compound C, Fig. 7) is an affinity label of MCAD with saturable rates of inactivation, strong substrate protection and irreversible inhibition of the enzyme [108]. Again the catalytic base, Glu376, reacts with this class of inhibitory thioesters probably via a simple nucleophilic displacement.…”

Section: Protein‐directed Inactivatorsmentioning

confidence: 99%

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Acyl‐CoA dehydrogenases

Ghisla

Thorpe

2004

European Journal of Biochemistry

307

297

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Acyl-CoA dehydrogenases constitute a family of flavoproteins that catalyze the a,b-dehydrogenation of fatty acid acyl-CoA conjugates. While they differ widely in their specificity, they share the same basic chemical mechanism of a,b-dehydrogenation. Medium chain acyl-CoA dehydrogenase is probably the best-studied member of the class and serves as a model for the study of catalytic mechanisms. Based on medium chain acyl-CoA dehydrogenase we discuss the main factors that bring about catalysis, promote specificity and determine the selective transfer of electrons to electron transferring flavoprotein. The mechanism of a,bdehydrogenation is viewed as a process in which the substrate aC-H and bC-H bonds are ruptured concertedly, the first hydrogen being removed by the active center base Glu376-COO -as an H + , the second being transferred as a hydride to the flavin N(5) position. Hereby the pK a of the substrate aC-H is lowered from > 20 to % 8 by the effect of specific hydrogen bonds. Concomitantly, the pK a of Glu376-COO -is also raised to 8-9 due to the decrease in polarity brought about by substrate binding. The kinetic sequence of medium chain acyl-CoA dehydrogenase is rather complex and involves several intermediates. A prominent one is the molecular complex of reduced enzyme with the enoyl-CoA product that is characterized by an intense charge transfer absorption and serves as the point of transfer of electrons to the electron transferring flavoprotein. These views are also discussed in the context of the accompanying paper on the three-dimensional properties of acyl-CoA dehydrogenases.

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Section: Protein‐directed Inactivatorsmentioning

confidence: 99%

“…Glu376 is the eventual target of this poorly understood oxidative inactivation reaction. Finally, certain 2‐halo‐acyl‐CoA compounds were found to be both substrates and inhibitors of the acyl‐CoA dehydrogenases [108,109]. For example, S ‐2‐bromohexanoyl‐CoA (compound C, Fig.…”

Section: Protein‐directed Inactivatorsmentioning

confidence: 99%

Acyl‐CoA dehydrogenases

Ghisla

Thorpe

2004

European Journal of Biochemistry

307

297

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“…It was further shown that the activity was attributed primarily to the ( S )-isomers. It was more recently shown that the shorter chain analogue 2-bromohexanoyl-CoA and the branched-chain analogue 2-bromo-4-methylpentanoyl-CoA act as both substrates and active site-directed inhibitors of the medium-chain acyl-CoA dehydrogenase …”

Section: Haloacyl-coenzyme a Thioestersmentioning

confidence: 99%

Coenzyme A Analogues and Derivatives: Synthesis and Applications as Mechanistic Probes of Coenzyme A Ester-Utilizing Enzymes

Mishra

Drueckhammer

2000

Chem. Rev.

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“…In addition to this work, GLU376 has been identified as a target of the mechanism-based inhibitor 2-octynoyl-CoA and the affinity label and alternate substrate 2-bromohexanoyl-CoA (Powell et al, 1988;Haeffner-Gormley et al, 1995). Proximity and orientation presumably preclude labeling of other potential amino acid side chains by these sitedirected inactivators of the medium-chain dehydrogenase.…”

Section: Discussionmentioning

confidence: 99%

Oxidative Inactivation of a Charge Transfer Complex in the Medium-Chain Acyl-CoA Dehydrogenase

Schaller¹,

Thorpe²

1995

Biochemistry

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The intense charge transfer complex between the enolate of 3-thia-octanoyl-CoA and the oxidized flavin of the medium-chain acyl-CoA dehydrogenase is discharged by the ferricenium ion with irreversible inactivation of the enzyme. Charge transfer complex formation is a necessary, but insufficient, condition for oxidative inactivation: the 3-oxa-octanoyl-CoA complex is also inactivated, whereas the comparable trans-3-octenoyl-CoA species is not. Complete inactivation of the dehydrogenase with 3-thia-octanoyl-CoA requires 1 molecule of thioester and apparently 3 molecules of ferricenium hexafluorophosphate. Experiments with 8-Cl-FAD substituted enzyme and the crystal structure of enzyme.ligand complexes argue that ferricenium ion-mediated oxidation proceeds through the flavin prosthetic group. Synthesis of [2-14C]-3-thia-octanoyl-CoA, followed by isolation of radiolabeled peptide from the modified medium-chain dehydrogenase, showed that inactivation results in labeling the catalytic base, GLU376. Oxidative modification is accompanied by the release of CoASH. A mechanism for inactivation is proposed involving generation of a sulfonium salt which efficiently captures the carboxylate nucleophile.

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S-2-Bromo-Acyl-CoA Analogs Are Affinity Labels for the Medium-Chain Acyl-CoA Dehydrogenase from Pig-Kidney

Cited by 5 publications

References 0 publications

Acyl‐CoA dehydrogenases

Acyl‐CoA dehydrogenases

Coenzyme A Analogues and Derivatives: Synthesis and Applications as Mechanistic Probes of Coenzyme A Ester-Utilizing Enzymes

Oxidative Inactivation of a Charge Transfer Complex in the Medium-Chain Acyl-CoA Dehydrogenase

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