QM/MM Analysis Suggests That Alkaline Phosphatase (AP) and Nucleotide Pyrophosphatase/Phosphodiesterase Slightly Tighten the Transition State for Phosphate Diester Hydrolysis Relative to Solution: Implication for Catalytic Promiscuity in the AP Superfamily

Abstract

Several members of the Alkaline Phosphatase (AP) superfamily exhibit a high level of catalytic proficiency and promiscuity in structurally similar active sites. A thorough characterization of the nature of transition state for different substrates in these enzymes is crucial for understanding the molecular mechanisms that govern those remarkable catalytic properties. In this work, we study the hydrolysis of a phosphate diester, MpNPP−, in solution, two experimentally well-characterized variants of AP (R166S AP, R166S/E322Y AP) and wild type Nucleotide pyrophosphatase/phosphodiesterase (NPP) by QM/MM calculations in which the QM method is an approximate density functional theory previously parameterized for phosphate hydrolysis (SCC-DFTBPR). The general agreements found between these calculations and available experimental data for both solution and enzymes support the use of SCC-DFTBPR/MM for a semi-quantitative analysis of the catalytic mechanism and nature of transition state in AP and NPP. Although phosphate diesters are cognate substrates for NPP but promiscuous substrates for AP, the calculations suggest that their hydrolysis reactions catalyzed by AP and NPP feature similar synchronous transition states that are slightly tighter in nature compared to that in solution, due in part to the geometry of the bimetallic zinc motif. Therefore, this study provides the first direct computational support to the hypothesis that enzymes in the AP superfamily catalyze cognate and promiscuous substrates via similar transition states to those in solution. Our calculations do not support the finding of recent QM/MM studies by López-Canut and coworkers, who suggested that the same diester substrate goes through a much looser transition state in NPP/AP than in solution, a result likely biased by the large structural distortion of the bimetallic zinc site in their simulations. Finally, our calculations for different phosphate diester orientations and phosphorothioate diesters highlight that the interpretation of thio-substitution experiments is not always straightforward.

Why nature really chose phosphate

“…That is, it was shown that the TS character is again dependent on the pK a of the leaving group, with a compact associative A N D N TS at high pK a that becomes progressively more expansive as the leaving group becomes more acidic (despite reproducing the LFER with chemical accuracy). Interestingly, this study covered a much smaller pK a range than that of the monoester dianions ; however, the overall correlation between leaving group pK a and mechanism appears to be similar, and qualitatively similar TSs have recently been obtained using QM/MM studies (Hou & Cui, 2012).…”

“…There have been a number of recent computational studies that have attempted to explore the issue of the comparative size of the TS for the solution and AP/NPP catalyzed hydrolysis of phosphate diesters (Hou & Cui, 2012 ;López-Canut et al 2010, 2011, which have obtained sharply contrasting results. López-Canut and co-workers (López-Canut et al 2010, 2011 have proposed that in both NPP and AP, phosphodiester hydrolysis proceeds through far more dissociative TS in the enzyme active site than in solution.…”

“…López-Canut and co-workers (López-Canut et al 2010, 2011 have proposed that in both NPP and AP, phosphodiester hydrolysis proceeds through far more dissociative TS in the enzyme active site than in solution. However, these works employed quite a low level of theory, and, as was also highlighted by (Hou & Cui, 2012), the Zn 2+ -Zn 2+ distance in these simulations increased to an unphysical value of as high as 7Á0 Å (Lopéz-Canut et al 2011), in sharp contrast to the crystal structure and also to data from Extended X-ray Absorption Fine Structure (EXAFS) and X-ray crystallography, which suggests little change in the binuclear Zn 2+ catalytic site during the course of the reactions catalyzed by AP and NPP (Bobyr et al 2012). In contrast to this, a more careful (but, as argued below, not yet fully reliable) study by (Hou & Cui, 2012) obtains a qualitatively similar phosphodiester TS in aqueous solution to that obtained by our earlier study (Rosta et al 2008), coupled with the more reasonable observation of a significant tightening of the TS in the enzyme active site (see also Luo et al 2012b).…”

“…Before moving the discussion on the above analysis, we may note that, although (Hou & Cui, 2012) recognizes the importance of validating their calculations by studies of the reference reaction in aqueous solution, the QM/PB calculation used is still not able to reproduce the experimental solution results. Furthermore, the correction by ab initio calculations both in the enzyme and solution are not determined at the ab initio least energy path.…”

“…Furthermore, the correction by ab initio calculations both in the enzyme and solution are not determined at the ab initio least energy path. The fact that the SCC-DFTBPR QM/MM approach is not fully calibrated on consistent solution surface, as well as the fact that the solution study is not done by the same approach as the enzyme calculation may in part explain why (Hou & Cui, 2012) could not reproduce the correct trend in the observed mutational effects. Additionally, it should be pointed out that, despite the fact that (Hou & Cui, 2012) claims that their calculations suggest that AP and NPP do not alter the nature of the TS relative to aqueous solution, their own data ( Fig.…”