Figure 4.

(a) Sequence alignment of AceK from several species. The DXDX(T/V) motif is highlighted in red. The preceding four hydrophobic residues are underlined, and the conserved lysine involved in transition state stabilization is shown in blue. (b) Structural alignment of AceK ⁴⁷⁵DXDX(I)⁴⁷⁹ motif (pink) with that of d,d-heptose 1,7-bisphosphate phosphatase [31] (PDB accession code: 3L8H; light blue). The yellow rectangle contains the four hydrophobic residues, and the strictly conserved lysine coordinated with Mg²⁺ ion. (c) Proposed phosphatase mechanism for AceK. (step 1) Asp475 acts as the nucleophile attacking the phosphate moeity on Ser113 of IDH through an associative mechanism to form a pentacovalent phosphate intermediate. (step 2) Asp477 acts as a general acid to donate its proton to Ser113 of IDH. The phosphoenzyme intermediate at Asp475 is formed. (step 3) The aspartatyl-phosphate undergoes nucleophilic attack by the ‘catalytic residue’ ADP to produce ATP. The breaking of the high-energy apartatyl-phosphate bond provides the energy for this reaction. (step 4) Water donates its proton to Asp477 and then attacks the γ-phosphate of ATP (ATP hydrolysis reaction). (step 5) The completed reaction with the enzyme available to perform its next round of reaction.