I studied the role of Coenzyme A in the ACS mechanism.
We thought that CoA was the probably last reactant to bind in the active site, for if it bound before the other two reactants (the CH3 and CO) then there must be some extraordinary mechanism to get those molecules in past the CoA.
It was also thought that the terminal sulfur of CoA bound directly to the nickel, since nickel-sulfur bonds are strong, and when the acetyl group bound the sulfur of CoA, the nickel unbound.
But thinking something is a far cry from proving it, and my research was to prove what CoA was doing in the active site.
We wanted to know two things:
Since CoA binding is not spectroscopically observable (believe me, we tried!) we had to resort to a very difficult method: equilibrium binding using radioactive CoA.
First, we needed CoA we could see, so I got some kinase (bacterial and porcine enzymes that attach phosphate groups onto small biological molecules like CoA) and some extremely hot radioactive phosphate (so hot the Geiger counter emitted a continuous tone). After the reaction I separated the phosphate from the hot CoA using HPLC (high-pressure liquid chromatography).
The binding study works like this:
Next: The Experiment
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