Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase

Abstract

Triosephosphate isomerase (TIM) is a proficient catalyst of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to d-glyceraldehyde phosphate (GAP), via general base catalysis by E165. Historically, this enzyme has been an extremely important model system for understanding the fundamentals of biological catalysis. TIM is activated through an energetically demanding conformational change, which helps position the side chains of two key hydrophobic residues (I170 and L230), over the carboxylate side chain of E165. This is critical both for creating a hydrophobic pocket for the catalytic base and for maintaining correct active site architecture. Truncation of these residues to alanine causes significant falloffs in TIM’s catalytic activity, but experiments have failed to provide a full description of the action of this clamp in promoting substrate deprotonation. We perform here detailed empirical valence bond calculations of the TIM-catalyzed deprotonation of DHAP and GAP by both wild-type TIM and its I170A, L230A, and I170A/L230A mutants, obtaining exceptional quantitative agreement with experiment. Our calculations provide a linear free energy relationship, with slope 0.8, between the activation barriers and Gibbs free energies for these TIM-catalyzed reactions. We conclude that these clamping side chains minimize the Gibbs free energy for substrate deprotonation, and that the effects on reaction driving force are largely expressed at the transition state for proton transfer. Our combined analysis of previous experimental and current computational results allows us to provide an overview of the breakdown of ground-state and transition state effects in enzyme catalysis in unprecedented detail, providing a molecular description of the operation of a hydrophobic clamp in triosephosphate isomerase.

Empirical Valence Bond Simulations Suggest a Direct Hydride Transfer Mechanism for Human Diamine Oxidase

“… Electrostatic contributions of the most significant residues (with a contribution >1 kcal·mol –1 ) to the calculated activation free energies (ΔΔ G elec,RS→TS ⧧ ) of the h DAO-catalyzed direct hydride transfer reaction. The depicted values were obtained by applying the linear response approximation to the calculated EVB trajectories and scaled by assuming an active site dielectric constant of 4, as in our previous work (ref ( 49 )). …”

Protein Flexibility and Stiffness Enable Efficient Enzymatic Catalysis

“…Many results are consistent with the conclusion that the structures for reactive Michaelis complexes of enzyme catalysts are stiff and allow for minimal protein motions away from highly organized forms. As noted above, enzyme-ligand complexes from X-ray crystallographic analyses serve as good starting points for calculations that model the experimental activation barrier for turnover at enzyme active sites, 14 , 15 so that the stiffness of reactive enzyme–substrate complexes is similar to that for crystalline enzymes. The empirical valence bond (EVB) computational methods developed by Arieh Warshel strongly emphasize the modeling of electrostatic interactions.…”

Epoxide hydrolysis as a model system for understanding flux through a branched reaction scheme

“…As EVB is a parameterized approach, the quality of the results provided by this approach depends on two things: (i) the quality of the parameterization used in the work (and thus the resulting EVB potentials) and (ii) the amount of conformational sampling performed and whether this is sufficient to obtain convergent results. A well parameterized EVB potential with adequate conformational sampling can easily give errors of less than 1 kcal mol À1 on the calculated energetics compared with experiment (see, for example, Barrozo et al, 2015;Blaha-Nelson et al, 2017;Kulkarni et al, 2017). In the case of the computational data presented in this work, obtaining reliable absolute energies for each enantiomer through calculations is difficult owing to the uncertainities in the energetics for the corresponding uncatalyzed reaction in aqueous solution, as also discussed in Amrein et al (2015) and Bauer et al (2016), hence the larger deviations between the experimental and calculated values.…”