Water and ion binding around RNA and DNA (C,G) oligomers

The biosynthesis of shikimate metabolites

Natural enzymes have arisen over millions of years by the gradual process of Darwinian evolution. The fundamental steps of evolution-mutation, selection, and amplification-can also be exploited in the laboratory to create and characterize protein catalysts on a human timescale. In vivo genetic selection strategies enable the exhaustive analysis of protein libraries with 10(10) different members, and even larger ensembles can be studied with in vitro methods. Evolutionary approaches can consequently yield statistically meaningful insight into the complex and often subtle interactions that influence protein folding, structure, and catalytic mechanism. Such methods are also being used increasingly as an adjunct to design, thus providing access to novel proteins with tailored catalytic activities and selectivities.

Investigating and Engineering Enzymes by Genetic Selection.

We present a density functional theory (DFT) hybrid quantum mechanical/molecular mechanical (QM-MM) implementation developed for simulations of reactions in complex environments. It is particularly suited to study enzyme active sites or solutes in condensed phases. The method combines a QM description of the solute with a MM treatment of the environment. The QM fragment is treated using DFT as implemented in the computationally efficient program SIESTA, while the environment is treated using the Wang et al. Amber force field parametrization. We applied our new QM-MM scheme to study the conversion of chorismate to prephenate by computing the reaction energy profile in vacuo, aqueous solution and in the active site of the B. subtilis chorismate mutase enzyme. We have performed calculations for two different choices of the QM subsystem in the enzyme simulations: including only the substrate moiety and the substrate plus the charged side chains glu78 and arg90, respectively. In both cases, our results are in good agreement with experiment. The catalytic activity achieved by chorismate mutase relative to the uncatalyzed reaction in solution is due to both a minor destabilization of the substrate molecule by compression and a major electrostatic stabilization of the transition state, which reduce the activation energy of the reaction.

A DFT-Based QM-MM Approach Designed for the Treatment of Large Molecular Systems: Application to Chorismate Mutase

A novel technique for computing free energy profiles in enzymatic reactions using the multiple steering molecular dynamics approach in the context of an efficient QM−MM density functional scheme is presented. The conversion reaction of chorismate to prephenate catalyzed by the Bacillus subtilis enzyme chorismate mutase has been chosen as an illustrative example.

Multiple-steering QM-MM calculation of the free energy profile in chorismate mutase.

A thermodynamic investigation of the Claisen rearrangement of chorismate2-(aq) to prephenate2-(aq) has been performed using microcalorimetry and high-performance liquid chromatography. The study used a well-characterized monofunctional chorismate mutase from Bacillus subtilis that was devoid of prephenate dehydrogenase and prephenate dehydratase activities. The calorimetric measurements led to a standard molar enthalpy change = −(55.4 ± 2.3) kJ mol-1 at 298.15 K for this reaction. An estimated value of the standard molar entropy change = 3 J K-1 mol-1 for the above reaction was used together with the experimental value of to obtain a standard molar Gibbs free energy change ≈ −56 kJ mol-1 and an equilibrium constant K ≈ 7 × 109 for the conversion of chorismate2-(aq) to prephenate2-(aq) at 298.15 K. Thus, for all practical purposes, this reaction can be considered to be “irreversible”. Quantum mechanics (Gaussian 94 with a B3LYP functional and a 6-31G* basis set) was used to calculate values of absolute and...

Thermodynamics of the Conversion of Chorismate to Prephenate: Experimental Results and Theoretical Predictions

A thermodynamic investigation of the conversion of chorismate2-(aq) to {pyruvate-(aq) + 4-hydroxybenzoate-(aq)} has been performed by using microcalorimetry and high-performance liquid chromatography. The study used a genetically engineered sample of chorismate lyase that was prepared with the Escherichia coli ubiC gene. The calorimetric measurements led to a standard molar enthalpy change = −(144 ± 7) kJ mol-1 for this reaction at the temperature T = 298.15 K and ionic strength Im = 0. An estimated value of the standard molar entropy change = 222 J K-1 mol-1 for the above reaction was used together with the experimental value of to obtain a standard molar Gibbs free energy change ≈ −210 kJ mol-1 and an equilibrium constant K ≈ 1037 for the conversion of chorismate2-(aq) to {pyruvate-(aq) + 4-hydroxybenzoate-(aq)} at T = 298.15 K and Im = 0. Quantum mechanics (Gaussian 94 with a B3LYP functional and a 6-31G* basis set) was used to calculate values of absolute energies for the neutral and ionic species per...

Kendall N. Houk

Papers

Thermodynamics of the Conversion of Chorismate to Prephenate: Experimental Results and Theoretical Predictions

Thermodynamic and quantum chemical study of the conversion of chorismate to (pyruvate + 4-hydroxybenzoate)