Leaving Group Ability Observably Affects Transition State Structure in a Single Enzyme Active Site

Abstract

A reaction’s transition state (TS) structure plays a critical role in determining reactivity and has important implications for the design of catalysts, drugs, and other applications. Here we explore TS structure in the enzyme alkaline phosphatase using hybrid Quantum Mechanics/Molecular Mechanics simulations. We find that minor perturbations to the substrate have major effects on TS structure and the way the enzyme stabilizes the TS. Substrates with good leaving groups (LGs) have little cleavage of the phosphorus-LG bond at the TS while substrates with poor LGs have substantial cleavage of that bond. The results predict non-linear free energy relationships for a single rate-determining step, and substantial differences in kinetic isotope effects for different substrates; both trends were observed in previous experimental studies, although the original interpretations differed from the present model. Moreover, due to different degrees of phosphorous-LG bond cleavage at the TS for different substrates, the LG is stabilized by different interactions at the TS: while a poor LG is directly stabilized by an active site zinc ion, a good LG is mainly stabilized by active site water molecules. Our results demonstrate the considerable plasticity of TS structure and stabilization in enzymes. Furthermore, perturbations to reactivity that probe TS structure experimentally (i.e., substituent effects) may substantially perturb the TS they aim to probe, thus classical experimental approaches such as free energy relations should be interpreted with care.

QM/MM Analysis of Transition States and Transition State Analogues in Metalloenzymes

“…[113] In addition to illustrating some of the computational methods available to enzymologists, we provide insights into the ability of TSAs to mimic the actual TS of the enzyme and the scope and limits of what can be gleaned from experimental observations of TSAs in this enzyme.…”

“…Despite sensationalistic claims of “trapping” TSs,[23, 102] actual TSs are unstable points on a free energy surface and thus cannot be trapped. Their structures can, however, be obtained through computational methods and our recent study of TS structure in AP[113] allows us to compare TSAs with actual TSs.…”

“…The simulations were conducted in accordance with the general methods described in refs. [63, 64, 113]. The starting structure was the crystal structure with ${\text{PO}}_4^{3-}$ used above, and the ligand was changed to pNPP in silico for the simulations with that ligand.…”

“…To answer this computationally, we must examine models for the actual TS, which we have recently done using DFTB3/MM free energy simulations;[113] the models of the TS are qualitatively consistent with diverse experimental data that include free energy relations, mutation effects and kinetic isotope effects. We note that DFTB3 was required because the active site of AP is very solvent accessible and therefore adequate sampling is crucial to the proper modeling of the TS structure (cumulatively more than 10 ns is used for each substrate in the DFTB3/MM umbrella sampling simulations); the DFTB3 method was also calibrated by a set of relevant phosphoryl transfer reactions and pK a calculations against higher level calculations as well as relevant experimental data (see SI of ref.…”

“…We note that DFTB3 was required because the active site of AP is very solvent accessible and therefore adequate sampling is crucial to the proper modeling of the TS structure (cumulatively more than 10 ns is used for each substrate in the DFTB3/MM umbrella sampling simulations); the DFTB3 method was also calibrated by a set of relevant phosphoryl transfer reactions and pK a calculations against higher level calculations as well as relevant experimental data (see SI of ref. [113]). What we found in that study is that the structure of the TS depends strongly on the LG for both phosphate mono- and diesters.…”