Department of Chemistry, Università della Calabria, 87036 Arcavacata di Rende, Italy.
Chemistry. 2013 Feb 4;19(6):2185-92. doi: 10.1002/chem.201201943. Epub 2012 Dec 28.
To elucidate the working mechanism of the "broad substrate specificity" by the Pseudomonas aeruginosa aryl sulfatase (PAS) enzyme, we present here a full quantum chemical study performed at the density functional level. This enzyme is able to catalyze the hydrolysis of the original p-nitrophenyl-sulfate (PNPS) substrate and the promiscuous p-nitrophenyl-phosphate (PNPP) one with comparable reaction kinetics. Based on the obtained results, a multistep mechanism including activation of the nucleophile, the nucleophilic attack, and the cleavage of the S-O (P-O) bond is proposed. Regarding the phosphate monoester, the results indicate that only some steps of the promiscuous reaction are identical to those in the native process. Differences concern mainly the last step in which the His115 residue acts as a general base to accept the proton by the O atom of the FGly51 in the PNPS, whereas in PNPP, the Asp317 protonated residue works as a general acid to deliver a proton by a water molecule to the oxygen atom of the C-O bond. The shapes of the relative potential-energy surface (PES) are similar in the two examined cases but the rate-determining step is different (nucleophile attack vs. nucleophile activation). The influence of the dispersion contributions on the investigated reactions was also taken into account.
为了阐明铜绿假单胞菌芳基硫酸酯酶（PAS）酶“广泛的底物特异性”的工作机制，我们在此进行了一项全量子化学研究，该研究是在密度泛函理论水平上进行的。该酶能够催化原始对硝基苯硫酸盐（PNPS）底物和混杂的对硝基苯磷酸盐（PNPP）的水解，其反应动力学具有可比性。基于获得的结果，提出了一个多步骤的机制，包括亲核试剂的活化、亲核攻击和 S-O（P-O）键的断裂。对于磷酸单酯，结果表明混杂反应的仅一些步骤与天然过程中的相同。差异主要涉及最后一步，其中 His115 残基作为一个通用碱，通过 FGly51 上的 O 原子接受 PNPS 中的质子，而在 PNPP 中，Asp317 质子化残基作为一个广义酸，通过水分子将质子传递到 C-O 键的氧原子上。在这两种情况下，相对势能面（PES）的形状相似，但决速步骤不同（亲核攻击与亲核活化）。还考虑了色散贡献对所研究反应的影响。
