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:

• Was the sulfur of CoA binding the nickel atom?
• Was the CoA binding before or after the other reactants?

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:

• enzyme is placed into small membrane cells into which small molecules move freely, but the large enzyme molecules cannot. These cells are placed in larger containers of buffered water.
• Radioactive CoA is added to the buffer (outside the membrane cell) and the apparatus is sealed.
• During the next 24 hours the cells are gently shaken to enhance mixing
• The contents of the membrane cell and the buffer are sampled for radioactive activity.
• If the CoA has bound to the protein, there will be more radioactivity (a measure of the total CoA concentration) inside the membrane than in the buffer.
• If the radioactivity is the same, there is no binding.
• Data is plotted and a non-linear least-squares analysis done to determine how much CoA is bound to the protein.

Next: The Experiment

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Last modified: 09/12/03