Stabilization of different types of transition states in a single enzyme active site: QM/MM analysis of enzymes in the alkaline phosphatase superfamily

Abstract

The first step for the hydrolysis of a phosphate monoester (pNPP(2-)) in enzymes of the alkaline phosphatase (AP) superfamily, R166S AP and wild-type NPP, is studied using QM/MM simulations based on an approximate density functional theory (SCC-DFTBPR) and a recently introduced QM/MM interaction Hamiltonian. The calculations suggest that similar loose transition states are involved in both enzymes, despite the fact that phosphate monoesters are the cognate substrates for AP but promiscuous substrates for NPP. The computed loose transition states are clearly different from the more synchronous ones previously calculated for diester reactions in the same AP enzymes. Therefore, our results explicitly support the proposal that AP enzymes are able to recognize and stabilize different types of transition states in a single active site. Analysis of the structural features of computed transition states indicates that the plastic nature of the bimetallic site plays a minor role in accommodating multiple types of transition states and that the high degree of solvent accessibility of the AP active site also contributes to its ability to stabilize diverse transition-state structures without the need of causing large structural distortions of the bimetallic motif. The binding mode of the leaving group in the transition state highlights that vanadate may not always be an ideal transition state analog for loose phosphoryl transfer transition states.

Figure 1

Basic reaction mechanism of the AP enzymes and the substrates studied here and in previous work. (a) The first step of phosphate mono ester hydrolysis catalyzed by AP. (b–c) The phosphate monoester (p-NitroPhenylPhosphate, pNPP²⁻) and diester (Methyl p-NitroPhenyl Phosphate, MpNPP⁻) studied in this and previous work, respectively.

Figure 2

The active sites of Alkaline Phosphatase (AP) and Nucleotide PyrophosPhatase/ phos-phodiesterase (NPP) are generally similar, with a few distinct differences. (a) E. coli R166S AP active site. (b) Xac NPP active site. The cognate substrates for AP and NPP are phosphate mo-noesters and diesters, respectively.

Figure 3

Benchmark calculations for pNPP²⁻ in R166S AP. Key distances are labeled in Å. Numbers without parenthesis are obtained with M06/6-31+G**/MM optimization; those with parentheses are obtained by SCC-DFTBPR/MM optimization with KO scheme. Asp369, His370, and His412 are omitted for clarity. (a) The reactant state in R166S AP; (b) The transition state in R166S AP by adiabatic mapping; (c) The overlay of crystal structure with ${\text{PO}}_4^{3-}$ (colorful), M06/6-31+G**/MM optimized structures with pNPP²⁻ (blue) and MpNPP⁻ (yellow). Hydrogen atoms are omitted. For additional comparisons of DFT(M06 or B3LYP)/MM and SCC-DFTBPR/MM structures, see Supporting Information.

Figure 4

Potential of Mean Force (PMF) calculation results for pNPP²⁻ hydrolysis in R166S AP with SCC-DFTBPR/MM. Key distances are labeled in Å and energies are in kcal/mol. (a) PMF along the reaction coordinate with error bar included; (b) Changes of average key distances along the reaction coordinate; (c) A snapshot for the reactant state, with average key distances labeled; (d) A snapshot for the TS, with average key distances labeled. Asp369, His370, and His412 are omitted for clarity.

Figure 5

The leaving group (p-Nitrophenyl) adopts diverse orientations during PMF simulations for the hydrolysis of pNPP²⁻ in both (a) R166S AP and (b) NPP. The distributions of the dihedral angle (P-O1-C-C, see Fig.2 for labels) in all umbrella sampling windows are plotted.

Figure 6

Benchmark calculations for pNPP²⁻ in NPP. Key distances are labeled in Å. Numbers without parenthesis are obtained with M06/6-31+G**/MM optimization; those with parentheses are obtained by SCC-DFTBPR/MM optimization with KO scheme. (a) The reactant state in NPP; (b) The transition state in NPP by adiabatic mapping. Asp257, His258, and His363 are omitted for clarity. For additional comparisons of DFT(M06 or B3LYP)/MM and SCC-DFTBPR/MM structures, see Supporting Information.

Figure 7

Potential of Mean Force (PMF) calculation results for pNPP²⁻ hydrolysis in NPP with SCC-DFTBPR/MM. Key distances are labeled in Å and energies are in kcal/mol. (a) PMF along the reaction coordinate; (b) Changes of average key distances along the reaction coordinate; (c) A snapshot for the reactant state, with average key distances labeled; (d) A snapshot for the TS, with average key distances labeled. Asp257, His258, and His363 are omitted for clarity.