Differences in the Nature of the Phosphoryl Transfer Transition State in Protein Phosphatase 1 and Alkaline Phosphatase: Insights from QM Cluster Models

Abstract

Quantum mechanical (QM) cluster models are used to probe effects on the catalytic properties of protein phosphatase 1 (PP1) and alkaline phosphatase (AP) due to metal ions and active site residues. The calculations suggest that the phosphoryl transfer transition states in PP1 are synchronous in nature with a significant degree of P-O_lg cleavage, while those in AP are tighter with a modest degree of P-O_lg cleavage and a range of P-O_nuc formation. Similar to observations made in our recent work, a significant degree of cross talk between the forming and breaking P-O bonds complicates the interpretation of the Brønsted relation, especially in regard to AP for which the computed β_lg/β_EQ,lg value does not correlate with the degree of P-O_lg cleavage regardless of the metal ions in the active site. By comparison, the correlation between β_lg/β_EQ,lg and the P-O_lg bond order is more applicable to PP1, which generally exhibits less variation in the transition state than AP. Results for computational models with swapped metal ions between PP1 and AP suggest that the metal ions modulate both the nature of the transition state and the degrees of sensitivity of the transition state to the leaving group. In the reactant state, the degree of the scissile bond polarization is also different in the two enzymes, although this difference appears to be largely determined by the active site residues rather than the metal ions. Therefore, both the identity of the metal ion and the positioning of polar or charged residues in the active site contribute to the distinct catalytic characteristics of these enzymes. Several discrepancies observed between the QM cluster results and the available experimental data highlight the need for further QM/MM method developments for the quantitative analysis of metalloenzymes that contain open-shell transition metal ions.

Figure 1:

The Protein Phosphatase-1 (PP1) and Alkaline Phosphatase (AP) feature a similar bimetallic binding site for a broad class of phosphate substrates (see Scheme 1). Shown in the boxes are the QM cluster models for the active site used here to compare the reactivities of PP1 and AP.

Figure 2:

The optimized substrate P-O_lg bond distances in PP1 and AP, as compared to water; also shown are results for QM cluster models of PP1 and AP with swapped metal ions. The P-O_lg bond is polarized and thus longer in length in the enzyme active sites. For the optimization in water, the substrate is solvated with twenty explicit water molecules (see Fig. S3), with the bulk water descried with CPCM.

Figure 3:

Comparison of the tightness, P-O distances and fractional bond orders for optimized transition states structures in PP1-Mn and AP-Zn QM cluster models.

Figure 4:

Superposition of the transition state structures in PP1-Mn(purple)/Zn(green) and AP-Mn/Zn with pNPP (tan) and pMPP (cyan) as substrates: the metal ions plays a major role in modulating the sensitivity of the transition state structure to variation in the leaving group.

Figure 5:

Metal ion effects on the transition state structures in PP1 and AP using metal-swapped QM cluster models.

Figure 6:

Comparison of O_nuc and O_lg charges in the phosphoryl transfer transition states in PP1 and AP with different substrates and metal ion occupation.

Scheme 1:

Aryl Phosphate substrates for PP1 and AP studied in this work

Scheme 2:

Phosphate monoester hydrolysis through a concerted pathway: bond formation to the nucleophile and bond fission to the leaving group occur simultaneously but not necessarily synchronously.