Phosphate Hydrolysis by the Fe2-Ca3-Dependent Alkaline Phosphatase PhoX: Mechanistic Insights from DFT calculations

PhoX is a pentanuclear metalloenzyme that employs two ferric ions and three calcium ions to catalyze the hydrolysis of phosphomonoesters. On the basis of the X-ray structure of PhoX ( Science 2014 , 345 , 1170 - 1173 ), a model of the active site is designed, and quantum chemical calculations are used to investigate the reaction mechanism of this enzyme. The calculations support the experimental suggestion, in which the two high spin ferric ions interact in an antiferromagnetic fashion. The two step mechanism proposed by experimentalists has been investigated. The nucleophilic attack of a trinuclear bridging oxo group on the phosphorus center was calculated to be the first step, which is concomitant with the departure of the phenolate, which is stabilized by a calcium ion. The second step is a reverse attack by a water molecule activated by a calcium-bound hydroxide, leading to the regeneration of the bridging oxo group. The second step was calculated to have a barrier of 27.6 kcal/mol. The high barrier suggests that the alternative mechanism involving phosphate release directly from the active site seems to be more likely. All five metal ions are involved in the catalysis by stabilizing the pentacoordinated trigonal bipyramidal transition states.