Substrate and Transition State Binding in Alkaline Phosphatase Analyzed by Computation of Oxygen Isotope Effects

Abstract

Enzymes are powerful catalysts and a thorough understanding of the sources of their catalytic power will facilitate many medical and industrial applications. Here we have studied the catalytic mechanism of alkaline phosphatase (AP), which is one of the most catalytically proficient enzymes known. We have used quantum mechanics calculations and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to model a variety of isotope effects relevant to the reaction of AP. We have calculated equilibrium isotope effects (EIEs), binding isotope effects (BIEs), and kinetic isotope effects (KIEs) for a range of phosphate mono- and diester substrates. The results agree well with experimental values, but the model for the reaction’s transition state (TS) differs from the original interpretation of those experiments. Our model indicates that isotope effects on binding make important contributions to measured KIEs on V/K, which complicated interpretation of the measured values. Our results provide a detailed interpretation of the measured isotope effects and make predictions that can test the proposed model. The model indicates that the substrate is deformed in the ground state (GS) of the reaction and partially resembles the TS. The highly preorganized active site preferentially binds conformations that resemble the TS and not the GS, which induces the substrate to adapt to the enzyme, rather than the other way around—as with classic “induced fit” models. The preferential stabilization of the TS over the GS is what lowers the barrier to the chemical step.

Regulation and Plasticity of Catalysis in Enzymes: Insights from Analysis of Mechanochemical Coupling in Myosin

“…The DFTB3/CHARMM approach was parameterized recently for phosphoryl transfer reactions (the 3OB/OPhyd variant), 61 and found to provide a semi-quantitative description of phosphoryl transfer transition states in alkaline phosphatase, including trends in the transition state structure 63 for a series of substrates, and also primary and secondary kinetic isotope effects. 64 Therefore, we expect that the approach is able to capture key trends in ATP hydrolysis in different conformational states of myosin. The computational efficiency of DFTB3 makes it possible to conduct much more thorough conformational sampling (by a factor of ~ 100) than previous DFT/MM studies.…”

Analysis of Phosphoryl-Transfer Enzymes with QM/MM Free Energy Simulations

“…The computational efficiency of the DFTB3/MM approach allowed us to compute the 18 O KIEs (Roston & Cui, 2016b) with both the bridging and nonbridging oxygen substitutions using a path-integral-based free energy perturbation approach (Major & Gao, 2007). The calculation assumes that the location of the transition state does not vary due to isotope substitution, while the vibrational contribution to the free energy is evaluated with anharmonicity included.…”

“…In fact, path integral calculations found rather significant binding isotope effects for the bridging oxygen in the ground state, suggesting that the substrate structure is deformed toward the transition state upon binding to the enzyme active site. For additional discussion, see Roston and Cui (2016b).…”

“…In recent years, as computational hardware and methodologies continue to improve, computational studies have become increasingly effective at analyzing the mechanism of reaction mechanisms in biomolecules. Specifically for phosphoryl-transfer reactions, hybrid quantum mechanics/molecular mechanics (QM/MM) type of computations (Brunk & Rothlisberger, 2015; Friesner & Guallar, 2005; Garcia-Viloca, Gao, Karplus, & Truhlar, 2004; Hu & Yang, 2008; Kamerlin, Haranczyk, & Warshel, 2009; Monard & Merz, 1999; Riccardi et al, 2006; Senn & Thiel, 2009) has been used by several research groups (Åqvist & Kamerlin, 2016; Carvalho, Szeler, Vavitsas, Åqvist, & Kamerlin, 2015; Duarte, Amrein, & Kamerlin, 2013; Genna, Vidossich, Ippoliti, Carloni, & De Vivo, 2016; Grigorenko et al, 2007; Hayashi et al, 2012; Hou & Cui, 2012, 2013; Kamerlin et al, 2013; Kiani & Fischer, 2014, 2016; McCullagh, Saunders, & Voth, 2014; McGrath, Kuo, Hayashi, & Takada, 2013; Mlynsky et al, 2014; Pabis, Duarte, & Kamerlin, 2016; Rosta, Kamerlin, & Warshel, 2008; Roston & Cui, 2016a, 2016b; Roston, Demapan, & Cui, 2016) to provide mechanistic insights into a broad set of enzymes that catalyze different phosphoryl transfers. These studies underlined subtleties in the interpretation of experimental data, which include both “direct” observations such as crystal structures and “indirect” observables such as kinetic isotope effects (KIEs), activation entropy, and (linear) free energy relationships.…”

Common mechanisms of catalysis in small and heterotrimeric GTPases and their respective GAPs

“…The subsequent reaction profile is only valid for the Ras · GAP system. Formally, this situation corresponds to a preferential binding of the transition state over the ground state within the protein (Roston and Cui, 2016). turnover assays; for example, the influence of site-directed mutants on the hydrolysis reaction cannot be studied.…”