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Aconitase: its source of catalytic protons.
1Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Biochemistry
|December 1, 1987
Summary
Aconitase enzyme catalysis involves more than one proton transfer during cis-aconitate hydration. This suggests a stabilized proton pool at the enzyme's active site.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Isotope effects
Background:
- Aconitase catalyzes the interconversion of citrate, isocitrate, and cis-aconitate.
- Proton transfer mechanisms in enzymatic reactions are crucial for understanding catalytic cycles.
Purpose of the Study:
- To investigate the proton dissociation rate from the donor site during cis-aconitate hydration by aconitase.
- To determine the number of enzyme-bound protons involved in the catalytic process.
Main Methods:
- Isotope partition experiments using tritium ([3H]) labeled water.
- Rapid mixing techniques to study enzyme kinetics.
- Analysis of isotope incorporation into citrate and isocitrate products.
Main Results:
- Citrate and isocitrate formed within 2 seconds showed higher tritium labeling than expected for a single proton transfer.
- Isotope incorporation exceeded four proton equivalents, indicating multiple proton involvement.
- The half-time for proton exchange with the solvent was approximately 0.1 seconds at 0°C.
Conclusions:
- Aconitase catalysis of cis-aconitate hydration involves a structurally stabilized pool of protons or water at the active site.
- This proton pool facilitates rapid proton exchange between the enzyme active site and the solvent.
- Proton transfer during citrate dehydration to isocitrate can occur directly or via the proton pool.