Fumarase - Mechanism

Mechanism

Figure 2 depicts the fumarase reaction mechanism. Two acid-base groups catalyze proton transfer, and the ionization state of these groups is in part defined by two forms of the enzyme E₁ and E₂. In E₁, the groups exist in an internally neutralized A-H/B: state, while in E₂, they occur in a zwitterionic A-/BH+ state. E₁ binds fumarate and facilitates its tansformation into malate, and E₂ binds malate and facilitates its transformation into fumarate. The two forms must undergo isomerization with each catalytic turnover.

Despite its biological significance, the reaction mechanism of fumarase is not completely understood. The reaction itself can be monitored in either direction; however, it is the formation of fumarate from S-malate in particular that is less understood due to the high pK_a value of the HR (Fig. 1) atom that is removed without the aid of any cofactors or coenzymes. However, the reaction from fumarate to L-malate is better understood, and involves a stereospecific hydration of fumarate to produce S-malate by trans-addition of a hydroxyl group and a hydrogen atom through a trans 1,4 addition of a hydroxyl group. Early research into this reaction suggested that the formation of fumarate from S-malate involved dehydration of malate to a carbocationic intermediate, which then loses the alpha proton to form fumarate. This led to the conclusion that in the formation of S-Malate from fumarate E1 elimination, protonation of fumarate to the carbocation was followed by the additional of a hydroxyl group from H₂O. However, more recent trials have provided evidence that the mechanism actually takes place through an acid-base catalyzed elimination by means of a carbanionic intermediate E1CB elimination (Figure 2).