Question

In reference to the citrate synthesis mechanism:

does the citrate synthase enzyme use acid catalysis, base catalysis, covalent catalysis or maybe a combo of these processes?

Answer 1

Citrate synthase is an enzyme utilized in Kreb's cycle. It catalyzes the process of citrate synthesis by the condensation of acetyl CoA and oxaloacetate.

acetyl-CoA + oxaloacetate + H2O → citrate + CoA-SH

It does this so by the acid-base catalysis. The chemical reaction involving the acid-base catalysis is accelerated by the addition of a acid or base. The main role of enzyme involved in such catalysis is proton transfer. The acid or base can donate and accept protons respectively to stabilize the charges in the developing transistion state which leads to activation of nucleophile and electrophile groups. Another characteristic of such catalysis reaction is the involvement of histinde as it has a pKa close to neutral which allows it to act as both an acid and a base.

The citrate synthase enzyme contains 2 active sites for binding of oxaloacetate and Co enzyme A and is composed of 3 amino acid residues: His274, His320, and Asp375. For the acid base catalysis the enzymes are designed to operate near the neutral pH and as you can see the citrate synthase contains histdine residue in its active site therefore it can be regulated at the pH 7. The enzymes that are involved in the acid base catalysis mainly activate the substrate by the addition or removal of a proton and the specific enzyme (citrate synthase ) also does this by deprotonation of acteyl CoA substrate. That is why the citrate synthase enzyme catalyses the citrate synthesis through acid-base synthesis.

The enzyme catalyzes by:

Deprotonation of acetyl CoA: For acid base catalysis procedure of the enzyme, it requires a nucleophile and an electrophile. The carbonyl group of oxaloacetate act as a n electrophile while the methyl group of acetyl CoA is activated into a nucleophile by deprotonation by Asp-375. The acetyl CoA in result is converted to form an enolate anions which is then neutralized by protonation through His-274 residue of the enzyme. This leads to the formation enol intermediate. A lone pair of electrons on His-274 formed due to the formation of enol intermediate accepts a proton to reform as enol anion. This results in the His-274 enol anion to initiate the nucleophilic attack on the carbonyl atom of oxaloacetate which in turn deprotonate the epsilon nitrogen atom of His-320.

Nucleophilic attack: The nucleophilic addition on the carbonyl atom results in the formation of citroyl-CoA. This leads to the deprotonation of water molecule by His-320 and hydrolysis is initiated. Now the lone pairs on oxygen can attack the carbonyl carbon of citroyl-CoA which leads to the release of -SCoA and the carbonyl stabilizes to reform. The −SCoA gets protonated to form HSCoA. And eventually, the hydroxyl which was added to the carbonyl is deprotonated and citrate is formed.