What question did this study set out to answer?

Determine the catalytic mechanism and the pathway of proton and hydroxyl transfer during aconitase-mediated interconversion of citrate and isocitrate.

April 1, 1967Journal of Biological ChemistryOpen Access

Mechanism of Aconitase Action

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Authors

IRIrwin A. RoseUniversity of California, Irvine EOEdward L. O'ConnellCancer Institute of Florida

Discussion

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Implication

In vitro enzymatic analysis reveals base-catalyzed proton abstraction in aconitase, indicating that substrate hydrogen transfers occur through a slowly dissociating conjugate acid.

Key Points

Determine the catalytic mechanism and the pathway of proton and hydroxyl transfer during aconitase-mediated interconversion of citrate and isocitrate.
Tracked hydrogen isotopes (tritium and deuterium) and oxygen-18 through intermediate steps of aconitase-catalyzed reactions.
Used coupled enzyme traps with isocitrate dehydrogenase and glutamate dehydrogenase to capture reaction intermediates and measure tritium incorporation into glutamate.
Assessed intramolecular versus intermolecular transfer pathways by varying cis-aconitate concentration and introducing tritiated 2-methyl hydroxy acid substrates.
Initial conversion of 3-tritiated isocitrate to citrate yielded tritiated citrate without loss of tritium, and deuterium transfer proceeded predominantly via an intramolecular route at baseline conditions.
Elevated cis-aconitate concentrations redirected substrate-derived tritium to isocitrate via intermolecular transfer, whereas hydroxyl groups labeled with oxygen-18 showed little to no transfer during hydroxy acid interconversion.
Substrate conversion proceeds through base-catalyzed proton abstraction, producing a slowly dissociating conjugate acid on the enzyme that donates the proton back to bound cis-aconitate before dissociation occurs.

Cite This Study

Rose et al. (1967) studied this question.

synapsesocial.com/papers/6a700adffebe604dd708d87a https://doi.org/10.1016/s0021-9258(18)96082-5

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April 1, 1967Journal of Biological ChemistryOpen Access

Mechanism of Aconitase Action

View Full Paper

Ask AI

Bookmark

Authors

IRIrwin A. RoseUniversity of California, Irvine EOEdward L. O'ConnellCancer Institute of Florida

Discussion

Member takes

Implication

In vitro enzymatic analysis reveals base-catalyzed proton abstraction in aconitase, indicating that substrate hydrogen transfers occur through a slowly dissociating conjugate acid.

Key Points

Determine the catalytic mechanism and the pathway of proton and hydroxyl transfer during aconitase-mediated interconversion of citrate and isocitrate.
Tracked hydrogen isotopes (tritium and deuterium) and oxygen-18 through intermediate steps of aconitase-catalyzed reactions.
Used coupled enzyme traps with isocitrate dehydrogenase and glutamate dehydrogenase to capture reaction intermediates and measure tritium incorporation into glutamate.
Assessed intramolecular versus intermolecular transfer pathways by varying cis-aconitate concentration and introducing tritiated 2-methyl hydroxy acid substrates.
Initial conversion of 3-tritiated isocitrate to citrate yielded tritiated citrate without loss of tritium, and deuterium transfer proceeded predominantly via an intramolecular route at baseline conditions.
Elevated cis-aconitate concentrations redirected substrate-derived tritium to isocitrate via intermolecular transfer, whereas hydroxyl groups labeled with oxygen-18 showed little to no transfer during hydroxy acid interconversion.
Substrate conversion proceeds through base-catalyzed proton abstraction, producing a slowly dissociating conjugate acid on the enzyme that donates the proton back to bound cis-aconitate before dissociation occurs.

Cite This Study

Rose et al. (1967) studied this question.

synapsesocial.com/papers/6a700adffebe604dd708d87a https://doi.org/10.1016/s0021-9258(18)96082-5

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