Citrate synthase catalyzes an aldo - type condensation. the kinetic mechanism is
ID: 809366 • Letter: C
Question
Citrate synthase catalyzes an aldo - type condensation. the kinetic mechanism is a random-sequential one where either substrate can bind first.
After reading about this reaction a student decided to do sme isotop exchange. When he incubated acoA with the enzyme in D2O he found no exchange of the methyl hydrogens with deuterium even after incubating for several days. However if he added succinic acid, along with AcCoA to the enzyme in D2O he found rapid exchange of the methyl hydrogens with the solvent deuteriums. What explanation for these results can give A1?
Explanation / Answer
Hi,
As you are aware the initiating step of the citric acid cycle occurs when a four carbon compound ( succinic acid) condenses with acetyl CoA (2 carbons) to form citric acid (6 carbons).
Now, Succinic acid can be easily deuterared with D2O as compared to Acetyl COA.
Check out the following link on deuterated succinic acid
http://www.sciencedirect.com/science/article/pii/0022236479902336
If you want to prepare deuterated acety CO-A, then you can follow the following steps
In first step, Coenzyme A (25 mg, 0.0325 mmol) should be added to 2 mL of ice-cold water. D3-acetic anhydride, 20uL was added with shaking and vortexing. The solution has a pH of approximately 5 at that step. After 20 min at 0OC, another 20uL of D3-acetic anhydride should be added, vortexed and the mixture was kept on ice for 30 min. Finally, the pH should be lowered to, 2 by addition of 10 mL of formic acid.