Question

d) e) f) Could 2,2,2-trifluoroacetyl CoA be processed by citrate synthase? Could 2-fluoroacetyl CoA be processed by citrate synthase? What is the mechanism of citrate synthase?

Answer 1

For understanding the substrate compatibility of the enzyme citrate synthase, first the enzyme's mechanism should be understood.

f) The enzyme has three key amino acids that catalyze the process - 2 histidines and 1 aspartate. Such a trio of amino acids that are responsible for the catalytic activity of an enzyme are called catalytic triads.

The general equation for the reaction catalyzed by the enzyme is: acetyl-CoA + oxaloacetate + H₂O → citrate + CoA-SH.

This achieved by the carboxylate of Asp deprotonating the alpha carbon Acetyl-CoA to give a stabilized carbanion that is protonated by an acidic proton of one His. The enol is then deprotonated at the hydroxyl group by another His' lone pair on the nitrogen, forming an enolate. This enolate then acts as a nucleophile that attacks oxaloacetate's carbonyl carbon forming citroyl-CoA while also deprotonating the protonated His thereby restoring catalytic activity. Simultaneously, a water molecule is deprotonated by a His and the hydrolysis of citroyl-CoA occurs to give citrate and CoA-SH.

A condensed mechanism shown below, from Wikimedia.

d) From the mechanism, it can be seen that the deprotonation of acetyl-CoA is a vital step that initiates the entire process. In 2,2,2-trifluoroacetyl-CoA, there are no hydrogens attached to the alpha-carbon of the molecule, thereby effectively prevents the entire reaction. So the molecule cannot process 2,2,2-trifluoroacetyl-CoA.

e) 2-fluoroacetyl-CoA has two hydrogen atoms attached to the alpha-carbon just as how acetyl-CoA has three. This makes the molecule a possible substrate that can be processed by the enzyme. Also, the presence of a strong electronegative F atom on the alpha-carbon stabilizes the negative charge on the carbanion, making it more stable than that of acetyl-CoA.