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Abstract

Phosphoserine phosphatase (PSP) utilizes one Mg²⁺ ion to catalyze the hydrolysis of phospho-l-serine. The displacement of Mg²⁺ by Ca²⁺ results in the loss of activity. The reaction mechanisms for the enzyme with both Mg²⁺ and Ca²⁺ bound were investigated using hybrid density functional theory. A large quantum chemical model abstracted from the X-ray crystal structure was employed in the calculations. Our calculations shed new insight into the catalytic mechanism of the natural enzyme and its lack of activity by Ca²⁺ substitution. For the catalytic reaction, our calculations showed that the whole reaction proceeds through two steps, namely dephosphorylation and phosphate hydrolysis. The associated barriers for these two steps are calculated to be 11.9 and 12.0 kcal mol⁻¹, respectively. The Mg-bound Asp11 residue functions as a nucleophile to attack the phosphorus moiety, in concomitant with the departure of the leaving group, which takes a proton from the neutral Asp13 residue. In the subsequent step, the newly formed anionic Asp13 residue activates a water molecule to perform the reverse attack on the phosphoryl intermediate, affording the phosphate product. The substitution of Mg²⁺ by Ca²⁺ results in different metal coordination fashion, in which the Asp167 residue changes from bidentate to monodentate and a second water molecule becomes ligated to Ca²⁺. The calculated barriers for the hydrolysis are ca 8 kcal mol⁻¹ higher than those in the native enzyme, which reconciles with the fact that Ca²⁺ inhibits the activity of PSP. Several possible reasons are discussed.