How is the active site of enolase organized to catalyze two different reaction steps?
Using a combined ab initio quantum mechanical/molecular mechanical approach developed in our laboratory, we obtained the reaction paths and free energy barriers for the two steps of the reaction catalyzed by enolase. In the first step, the α-proton of the substrate, 2-phospho-D-glycerate (PGA), is removed by Lys345, resulting in an enolic intermediate. In the second step, the β-hydroxyl group leaves the enolic intermediate with the assistance of a general acid, Glu211. The calculated free energies of activation are 13.1 and 9.4 kcal mol for the first and the second step, respectively. The barrier heights are consistent with the reaction rates measured from experiments. The calculations indicate that the electrostatic interactions between the substrate and two divalent metal cations at the active site strongly favor the first step. However, the same metal cations strongly disfavor the second step because the change in charge of the substrate is of an opposite sign compared with that in the first step. We conclude that the enzyme environment (excluding Lys345, Glu211, and the metal cations) forms an essential part of the reaction mechanism. It counterbalances the disfavoring effects of the metal cations in the second step without interfering with the first step despite the reversed charge changes of the substrate in the two steps. This capability of the enzyme originates from the three-dimensional organization of polar and charged groups in the active site of enolase, as indicated by correlations between the three-dimensional structure and energetic analyses based on our calculations. To achieve overall catalytic efficiency, the structure of the enolase active site takes advantage of the fact that the charge reorganization procedures accompanying the two reaction steps take place in two different directions in space. -1
Liu, H; Zhang, Y; Yang, W
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